WO2008103786A1 - Multi-strip western blotting procedure to increase quantitative data output - Google Patents

Abstract

The present invention relates to improved technique for comparative quantitative analyses of large numbers of biological samples such as proteins and nucleic acids. The invention relates to increased and reliable data output when multiple proteins are studied at the same time. The improve technique involves assembling a unique multi-strip gel for electro-transfer. Methods and devices are provided for the cutting and assembling the multi-strip gel.

Description

MULTI-STRIP WESTERN BLOTTING PROCEDURE TO INCREASE QUANTITATIVE DATA OUTPUT

CROSS REFERENCE TO RELATED APPLICATIONS

[01 ] This application claims the benefit under 35 U. S. C. § 119(e) of U.S. provisional application No. 60/903,152 filed February, 23 2007, the contents of which are incorporated herein by reference in its entirety.

GOVERNMENT SUPPORT

[02] This invention was made with Government support under GM59570 and AAOl 15311 awarded by the US National Institutes of Health. The government has certain rights in this invention.

BACKGROUND OF THE INVENTION

[03] The qualitative and quantitative measurement of protein abundance is one of the common tasks in biomedical diagnostics in the search for autoantigens, biomarkers and in identifying such diseases as syphilis [1, 2], human immunodeficiency virus [3-7], Lyme disease [8, 9], various autoimmune disorders [10-15], Creutzfeldt-Jakob disease [16-19], cancer [20-24] and many others [25-27].

[04] Genomic methods (microarrays, PCR, SNP analysis) have many advantages, but do not directly measure the dynamics or functional state of cellular proteins. The quantitative measurement of protein modification states in response to cell stimulation, the identification of specific protein-protein interactions, establishing the contribution of unique proteins to the downstream processing of signals, and analysis of differences in protein expression levels among various cell types, is essential for understanding protein networks involved in signal transduction, linking proteins of unknown function to known cellular processes and unraveling the molecular mechanisms of specific signaling pathways and regulation of cell fate. In systems biology, the experimental data can be used as a basis to build a comprehensive mathematical model of the system of interest [28-32]. High-quality signal and accurate quantitation of data is essential for constructing informative models of the cellular signaling system. [05] In studies of cell signaling the degree of protein phosphorylation in stimulated cells is compared with the basal phosphorylation level of the same protein (control). Often, the predictions of mathematical models may demand experimental data to be generated in the presence of one or more cellular perturbations, e.g. pharmacological inhibitors, exposure to physiochemical stresses or the downregulation of protein expression). Additionally variations in the dose and strength of a stimulus, measurement of detailed protein activation kinetics versus a single time-point and protein localization provide deeper insight into the threshold-dependant spatio-temporal functioning of a specific cell signaling pathway [33]. [06] Hence, these tasks require producing large amounts of reproducible high-quality quantitative data with low variance. The processing of such high-throughput data is a costly, time-consuming multi-step procedure prone to systematic or random errors. Therefore improvements to existing experimental methods are desirable, which provide cheaper, faster and better detection of proteins [7, 34-40].

[07] The Western blotting technique for the immunodetection of the expression levels and phosphorylation status of electrophoretically resolved proteins [41-45] is widespread, but has several drawbacks. In a traditional Western blotting technique, each protein gel is used in its entirety during the Western blotting transfer process. Therefore all separated proteins on the gel, regardless of their molecular sizes, post-translational modifications such as phosphorylation or glycosylation status, and the quantity, will experience the same Western blotting transfer conditions.

[08] However it is known that larger sized proteins (>100 kilo Daltons) are harder to transfer completely from the gel on to the Western blot membrane and as such usually require more Western transfer time and/or slight changes to the buffer system used during the transfer. Conversely, smaller sized proteins transfer to Western blot membranes in less amount of time. So a standard Western transfer time will result in complete transfer of the proteins < 100 kilo Daltons, but incomplete transfer for those proteins > 100 kilo Daltons. [09] Under the one-protein-gel-transfer-to-one-blot situation, it is not possible to quantitatively analyze multiple proteins simultaneously. In order to quantitatively compare many distinct proteins from a large number of samples obtained under various experimental conditions or independent experiments, the scientist has to run many protein gels and transfer them on to many membrane blots for analyses. The greater number of different proteins to be analyzed, the more protein gels and blots needed because one-protein-gel-to-blot can be used for quantifying only one protein accurately. For example if there are five distinct proteins to study for all the samples, at least five identical set of protein gels and corresponding membrane blots are needed. Cumulative variations in protein gel separation and Western blotting transfer conditions often contribute significantly to the standard deviations in the final data obtained from the Western blotting technique and makes it difficult to compare the data quantitatively.

SUMMARY OF THE INVENTION

[010] The invention relates to an improved technique for comparative quantitative analyses of a large number of protein samples. More particularly, the invention relates to the increased and reliable data output when multiple proteins are studied at the same time and the proteins are derived from large numbers of samples collected under different experimental conditions and independent experiments. The invention provides a method of obtaining large amount of highly reproducible and quantitative data output and devices for the implementing the method.

[01 1 ] In one embodiment, the invention provides a method for increasing quantitative data output of biological molecules from multiple samples comprising: (a) performing gel electrophoreses on a plurality of same sized gels; (b) electrophoretically resolving each gel under conditions to result in substantially identical separation of molecular markers on the gels;

(c) dividing each gels into strips that correspond to a set of pre-determined zones based on the location of the molecular marker on the gels; and (d) the strips from equivalent zone from each gel in parallel tandem with each other and in contact with a filter paper to form a multi-strip gel.

The strips from equivalent zones are, for example, all the zone 1 gel strips or all the zone 2+3 gel strips of the plurality of cut gels.

[012] In one embodiment, the multi-strip gel is assembled with more than two gel strips from the equivalent zones in different gels. A multi-strip gel can have up to 10 gel strips from ten different gels. In another embodiment, the multi-strip gel is assembled with more than two gel strips from the equivalent zones in different gels and more than two gel strips from another equivalent zone in different gels.

[013] In another embodiment, the gel strips can cover two zones instead of one zone. In another embodiment, the multi-strip gel can be assembled from several gel strip from one zone and several gel strip from another gel zone. In addition, a single multi-strip gel can comprise gel strips from different zones, for example, three gel strips from zone 1 and three gel strips from zone 5.

[014] In one embodiment, any biological molecule that can be separated electrophoreticaly is considered for the method disclosed. Examples include protein, RNA, or DNA.

[015] The plurality of same sized gels can be made of polyacrylamide or agarose. The gels can be casted manually or are precast gels. The precast gels can be PreCut'n'Cast gels which are gels that have pre-cut zones within the gel in the gel cassette. This eliminates the need to cut the gel after separating the biological molecules on the gel.

[016] In addition, the plurality of same sized gels can be gradient polyacrylamide gels, single percentage polyacrylamide gels, and the second dimensional vertical slab polyacrylamide gels of two-dimensional gel electrophoresis.

[017] The dimensions of the same sized gels that can be use with the present invention include the following: (a) mini gels of 8 X 8 cm; (b) mini gels of 8.6 X 6.8 cm; (c) midi gels of 13 X 8.3 cm; (d) mini gels of 9 X 6 cm; (e) mini gels of 10.5 X 6 cm; (f) mini gels of 12.5 X 6 cm; and (g) large gels of 20 x 20 cm

[018] In one embodiment, the number of same sized gels can be from 2 to 12.

[019] In one embodiment, the molecular markers used in the invention are pre-stained. These pre-stained markers are visible to the naked eye and allow the scientist to determine the location, on the gel, of the biological molecule with approximately the same size as a specific molecular marker. With that estimation, the scientist can divide the gel into the correct number of zones so as the zones encompass the biological molecule of interest.

[020] The method described further includes electro-transferring the multi-strip gel on to a membrane, and the electro-transfer membrane is nitrocellulose, PVDF, or nylon. The resultant the membrane is further processed for Western blot analysis and protein quantification.

[021 ] In one embodiment, the multi-strip gel is dried. The dried multi-strip gel is then exposed to autoradiograph films or storage phosphor screens.

[022] In another embodiment, the multi-strip gel is stained with nucleic acid stains.

[023] In another embodiment, devices designed to facilitate the gel cutting and assembly of the multi-strip gels are provided.

BRIEF DESCRIPTION OF FIGURES

[024] Figure 1 shows that the non homogeneous gel and separate transfer conditions cause significant signal variability.

[025] Figure 2A is a schematic diagram showing an example of the designation of multiple gels that are utilized in the multi-strip Western blotting procedure.

[026] Figure 2B shows the identification and cutting of the protein migration zones in the Multi-strip Western blotting procedure. M, prestained protein molecular weight marker; BDF, blue dye front; H, the distance from BDF to the center of particular marker band. Scissor symbols indicate the cutting lines, which would separate the entire gel into nine strips. Protein migration zones are enumerated by numbers in circles.

[027] Figure 2C shows a schematic diagram of an assembled multi-strip western blot comprising solely of the zone 1 gel strips from the protein gels A, B, C, D, E, and F. [028] Figure 2D shows a schematic diagram of an assembled multi-strip western blot comprising solely of the zone 2+3 gel strips from the protein gels A, B, C, D, E, and F. [029] Figure 2E shows a schematic diagram of an assembled multi-strip western blot comprising the zone 4+5 gel strips and the zone 6 gel strips from the protein gels A, B, and C. The blot can be cut at the scissor marker to give two blots, one blot with proteins from zone 4+5, and the other blot with proteins from zone 6. [030] Figure 2F shows a schematic diagram of an assembled multi-strip western blot comprising the zone 4 gel strips, the zone 5 gel strips, and the zone 6 gel strips from the protein gels A and B. This blot can be separated as indicated by the scissors markers.

[031 ] Figure 3 A shows the Western blot analyses of phospho-SHP2 (Tyr542) from ten replicates of zone 4 gel strips from ten independent protein gels A-J. Zone 4 is one of the upper panels in a protein gel.

[032] Figure 3B shows the Western blot analyses of phospho-ERK from ten replicates of zone 5 gel strips from ten independent protein gels A-J. Zone 5 is one of the middle panels in a protein gel.

[033] Figure 3C shows the Western blot analyses of GRB2 from ten replicates of zone 6 gel strips from ten independent protein gels A-J. Zone 6 is one of the bottom panels in a protein gel.

[034] Figure 4A shows the Western blot analyses of RasGAP from triplicates of zone 7 gel strips from three independent protein gels A-C. Zone 7 is one of the bottom panels in a protein gel.

[035] Figure 4B shows the Western blot analyses of phospho-SHP2 (Tyr542) and unphosphorylated SHP2 from replicates of zone 4 gel strips from four independent protein gels

A-D. Zone 4 is one of the upper panels in a protein gel.

[036] Figure 5A demonstrates that the signal variability associated with the multi-strip

Western blot analysis is markedly reduced compared to the conventional single gel Western blot analysis.

[037] Figure 5B shows histogram representations of signal variabilities associated with the multi-strip Western blot analyses compared to the conventional single gel Western blot analyses for several signaling proteins.

[038] Figure 6A shows the Western blot analyses of phospho-ERK (left panel) and GB R2

(right panel, used as control) from replicates of zone 5 gel strips and zone 6 gel strips respectively of seven independent protein gels A-G in a time-course EGF signaling stimulation experiment of HEK293 cells.

[039] Figure 6B shows the Western blot analyses of phospho-ERK (left panel, zone 5 replicates) and phospho-AKT and phospho-SHP2 (right panel, zone 4 replicates) from three independent protein gels A-C of a EGF signaling stimulation experiment of HEK293 cells in the presence and absence of the inhibitor wortmannin.

[040] Figure 7 A shows the Western blot analyses of phospho-AKT and D -tubulin (as gel loading control) from four synchronous cell types: HEK293, A549, T24, and MCFlOA, in a time-course stimulation experiment with EGF. [041 ] Figure 7B shows the Western Blot analyses of phospho-ERK2 (zone 4) from HEK293 and T24 cells stimulated with various amounts of IGF-I, EGF, and HGF. Non-phospho-ERK2

(aERK2) protein was added to the cell lysate loading sample prior to gel loading to establish the standard curve (zone 5).

[042] Figure 7C shows the standard curve of Western blot signal intensity of aERK2 obtained for Figure 7B.

[043] Figure 7D shows the raw intensity signal histogram (left) and the converted protein quantity histogram (right) obtained from the Western Blot analyses of phosphor-ERK2 from cell lysates of HEK293 and T24 cells stimulated with various amounts of IGF-I, EGF, and HGF.

[044] Figure 8A demonstrates some of the observed artifacts of protein bands in Western blotting.

[045] Figure 8B shows the comparison of protein band quality in old and fresh gels. Gels were used two months before and after their expiration date.

[046] Figure 9 demonstrates how the multi-strip Western Blot procedure can reduce the interference of non-specific bands by the removal of the gel strip containing the interfering band.

Non-specific bands is indicated by asterisk symbol. The desired gel area is now separated from the non-specific band.

[047] Figure 1OA shows a schematic diagram of a mini-slicer (top view). A-adjustable legs with lock and spring mechanism; B-channels for fixers that hold and fix the knives in place; C- ruler; D- knives; E- fixers that hold and fix the cutting knives in place.

[048] Figure 1OB shows a schematic diagram of the cross-section of the mini-slicer adjustable leg A, showing the UP position and DOWN position.

[049] Figure 1OC shows a schematic diagram of the lock mechanism to regulate the position of the knives in the channel.

[050] Figure 11 shows the major components and steps for multi-strips assembling by the auto-slicer.

[051 ] Figure 12A shows one possible variant of the PreCut'n'Cast gel with precut evenly sized gel strips (dashed lines) on a gel. This gel is designed to capture proteins of various molecular sizes in the middle of desired slices by varying the running time of electrophoresis.

[052] Figure 12B shows another possible variant of the PreCut'n'Cast gel with unevenly sized strips that is customized for capturing some signaling proteins of specific molecular weights from Table 1-9.

[053] Figure 12C shows another example of PreCut'n'Cast gel with unevenly sized strips that is customized to capture some signaling proteins of specific molecular weights from Table 1-9. [054] Figure 13 demonstrates two gel loading strategies when the number of samples exceeds the number of gel wells.

[055] Figure 14 shows an example of a single gel that can be divided into five (left panel, case 1) or four (right panel, case 2) strips containing distinct protein zones. The strips will be subsequently transferred onto assembling filter papers together with similar strips derived from other gels.

[056] Figure 15A shows an example of an assembly of multiple gel strips onto assembling filter paper (AFP). Five strips were cut out of each gel (gel A, B, C, D, E and F) and combined onto appropriate assembling filter papers AFP #1 through #6.

[057] Figure 15B shows another example of an assembly of multiple gel strips onto assembling filter paper (AFP). Four strips were cut out of each gel (gel A, B, and C) and combined onto two assembling filter papers, AFP #1 and #2.

[058] Figure 16. Comparison of protein migration patterns in Control and Pre-Cut polyacrylamide gels. Prestained Precision Plus Protein Standards (Bio-Rad) marker was separated by SDS-PAGE electrophoresis and photographed. Arrow indicates the cutting line in the gel.

DESCRIPTION OF THE INVENTION

[059] Unless otherwise defined herein, scientific and technical terms used in connection with the present application shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[060] It should be understood that this invention is not limited to the particular methodology, protocols, and reagents, etc., described herein and as such may vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the claims. [061 ] Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood as modified in all instances by the term "about." The term "about" when used in connection with percentages may mean ±1%.

[062] All patents and other publications identified are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that might be used in connection with the present invention. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicants and does not constitute any admission as to the correctness of the dates or contents of these documents.

[063] Unless otherwise stated, the present invention is performed using standard procedures, as described, for example in Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (1982); Sambrook et al., Molecular Cloning: A Laboratory Manual (2 ed.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y., USA (1989); Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (1986); Current Protocols in Protein Science (CPPS) (John E. Coligan, et. al., ed., John Wiley and Sons, Inc.) and Current Protocols in Immunology (CPI) (John E. Coligan, et. al., ed. John Wiley and Sons, Inc.), Current Protocols in Cell Biology (CPCB) (Juan S. Bonifacino et. al. ed., John Wiley and Sons, Inc.), Culture of Animal Cells: A Manual of Basic Technique by R. Ian Freshney, Publisher: Wiley-Liss; 5th edition (2005), Animal Cell Culture Methods (Methods in Cell Biology, VoI 57, Jennie P. Mather and David Barnes editors, Academic Press, 1st edition, 1998) which are all incorporated by reference herein in their entireties.

[064] The present invention is directed to a method for increasing quantitative data output of biological molecules from multiple samples. In one embodiment, the method comprises performing polyacrylamide gel electrophoresis on a plurality of same sized gels; electrophoretically resolving each gel under conditions to result in substantially identical separation of pre-stained markers on the gels; dividing each gel into multiple strips that correspond to a set of pre-determined zones on the gel based on protein molecular size; and arranging the equivalent zones from each gel in parallel tandem on a assembling filter paper so that the zones would contact a Western blot membrane to form a multi-strip gel-membrane sandwich ready for Western transfer.

[065] The present invention is suitable for increasing quantitative data analyses of biological molecules such as protein, RNA or DNA from multiple samples. The number of same sized gels can be from, for example, 2 to 12. The same sized gels can be commercially available precast gels that come in a variety of sizes and gel percentages. The protein mini gels are 8 X 8 cm (gel cassette size is 10 X 10 cm) or 8.6 X 6.8 cm (gel cassette size is 10 X 8 cm) and the protein midi gels are 13 X 8.3 cm. Large gels have cassette size of 20 x 20 cm. Examples of nucleic acid mini gel sizes are 9 X 6 cm, 10.5 X 6 cm, and 12.5 X 6 cm. Several manufacturers (Sigma, EPBox, Invitrogen, Cambrex, LifeGels, BioRad) offer such precast gels. [066] In one embodiment, the PreCut'n'Cast gels are used for the invention disclosed herein. PreCut'n'Cast gels are precast gels that are already divided into several pre-determined gel strips corresponding to several zones. No cutting of the gel is required after the biological sample have been separated. The pre-determined strips can be simply separated from the adjacent strips that make up the PreCut'n'Cast gel and be used to assemble the multi-strip gel. [067] In one embodiment, the invention is suitable for protein studies by Western blot analysis and protein quantification. Protein quantification following Western blot analysis can be conducted using conventional chemiluminescent, chemifluorescent, colorimetric chemistry. In another embodiment, the method is also suitable for nucleic acids studies. The RNAs and DNAs in a multi-strip gel can be stained with SYBR® Green EMSA nucleic acid gel stain and quantified by fluorescence using a fluoroimager such as Typhoon™, Storm™, or Fluoroimager by Molecular Dynamics.

[068] The terms electro-transfer and electroblotting refer to the use of electric current to transfer biological molecules from a electrophoresis gel on to a membrane such as nitrocellulose, polyvinylidene fluoride (PVDF), or nylon. Both terms are used interchangeably herein. The biological molecules include proteins, and nucleic acids such as RNA and DNA but are not limited to these.

[069] Western blot membranes include nitrocellulose, polyvinylidene difluoride (PVDF) and nylon, but are not limited to just these membranes.

[070] In one embodiment, the invention is a modified method of the traditional Western blotting technique. The modified Western blot technique substantially improves data accuracy by up to two-folds, and dramatically increases data output per one blotting cycle of up to ten- folds. It is based on simultaneous transfer of proteins from multiple gel strips on to the same membrane and is compatible with any conventional gel electrophoresis systems. Thus for protein analysis, this version of the present invention is termed the multi-strip Western blotting procedure. An apparatus for performing this invention is also provided. [071 ] In one embodiment, the invention is applicable to all known polyacrylamide gel electrophoresis systems including non-denatured (native) and denatured protein polyacrylamide gels where sodium dodecyl sulfate (SDS), lithium dodecyl sulfate (LDS), DL-dithiothreitol, and Dmercaptoethanol are the common detergent/denaturants, gradient and single percentage polyacrylamide gels, continuous and discontinous polyacrylamide gels, the second dimensional vertical slab polyacrylamide gels of two-dimensional gel electrophoresis systems, and also nucleic acids (DNA and RNA) polyacrylamide and agarose gels. The gel electrophoresis buffer systems can be the following commonly used buffer systems, but are not limited to these: Tris- glycine, Bis-Tris, Tricine-glycine, Tricine, Tris-glycine-glycerol, Tris-borate-EDTA, Tris- acetate-EDTA, Tris-acetate-EDTA-urea, and Tris-acetate.

[072] In one embodiment, the present invention allows simultaneous analyses of up to nine distinct proteins separated on a single 10 X 10 cm (gel cassette) mini gel. For vertically larger- sized gels, a larger number of distinct proteins can be electrophoretically separated and therefore a larger number of distinct proteins can be studies using the inventions disclosed herein, up to fifteen distinct proteins for a 20 X 20 cm (gel cassette) large protein gel. This invention eliminates the need to run many proteins gels in order to analyze more than one distinct protein of interest.

[073] The present invention allows the creation of a single Western blot containing all the same types of protein from many different samples that were separated on different protein gels. In this case, since all the same types of protein from many samples are assembled on to one multi-strip gel and undergo a single Western blot transfer, these proteins will experience identical transfer conditions, therefore the variations due to electro-transfers of multiple blots are eliminated.

[074] In accordance with one embodiment of the present method, after electrophoresis separation of the protein samples on protein gels, the protein gels are cut into several predetermined horizontal gel strips based on location of the molecular sizes of the pre-stained markers. In one embodiment of the method, the pre-stained molecular marker is BioRad's Precision Plus pre-stained marker. The horizontal strips from the same equivalent region on each gel contain the same types of protein based on separation by its molecular weight. [075] A molecular marker is a cocktail of several molecules of different molecular weight. For example, a protein molecular marker is made of several proteins of differing molecular weight, such as 10, 30, 50, 90, 150, 250 kilo Daltons (kDa). Similarly, a nucleic acid DNA marker is made of DNA fragments of differing length, such as, 500, 1000, 3500, 4900, 5400, 21000 base pairs. The protein or nucleic acid markers can be conjugated to an additional molecule to make the protein or nucleic acid visible to the naked eye. This makes them give pre-stained molecular markers. During electrophoresis, protein or nucleic acid molecules in a gel migrate and distribute in the gel on the basis of their sizes. Since nucleic acids and proteins are mainly colorless and invisible, the pre-stained molecular markers provide a guide to where proteins or nucleic acids of the corresponding molecular weight should be found approximately on the gel. Therefore, a gel strip can be selected for the region between 30-50 kDa in a protein gel. [076] In one embodiment, each strip corresponds to a zone on the protein gel. Strips from the equivalent zones in different gels are removed and aligned in rows in contact with a filter paper to form a multi-strip protein gel. After electro-transfer on to a Western blot membrane, a multi- strip Western blot is obtained and this blot will have horizontal rows of the same type of proteins obtained from many different samples that were electrophoretically separated on different gel. This single blot is then processed for immunoblotting analysis and protein quantification. [077] In another embodiment, the multi-strip protein or nucleic acid gel can be used directly for autoradiography of isotope-labeled molecule of interest. The multi-strip gel is dried and then exposed to traditional autoradiographic films (Kodak Inc.) or exposed to photon-sensitive screens such as the storage phosphor screens by GE Healthcare Life Science/ Molecular Dynamics or Fuji and then used in conjunction with the Typhoon™, Storm™, Phosphorlmager™ (Molecular Dynamics ) or Fuji phosphorimager.

[078] In one embodiment, the method of the invention is performed in the following example as described for protein samples. The method is equally applicable for nucleic acids such as DNA and RNA.

[079] In order to analyze a number of distinct proteins from a number of protein samples, run several same sized protein gels, for example 10 X 10 cm (gel cassette) mini gels, all gels with 10 wells each. For comparative quantitative analyses, each well should be loaded with equal amount of protein such as 20 μg total protein per well/lane (Fig. 2A).

[080] Load the designated lane 1 (from the left) with pre-stained markers such that there is at least 1 Dg of each of the molecular markers in lane 1 (Fig. 2A).

[081 ] Run the gels under the same conditions so that the dye front migrates to the same spot at the bottom of each gel in the end of electrophoresis. Proteins migrate downwards from the wells located at the top of the gel. Larger sized proteins migrate slower while smaller size proteins migrate faster. Thus at the end of protein electrophoresis, the larger sized proteins are separated from the smaller sized proteins (Fig. 2B).

[082] When the dye front has reached the bottom of the gel, turn off the electricity. [083] Remove the gel sandwich plates from the gel apparatus, pry open the sandwich and remove one gel plate, the one without the flexible gel attached; the remaining gel plate has the gel lying on the plate. The orientation of each gel on the glass plate should be such that the pre- stained markers are found on the first left lane and the dye front is at the bottom (Fig. 2B, enlarged). Gently lift and re-arrange the gel if the gel is in the improper orientation. If six protein gels are prepared, label the protein gels A to F (Fig. 2A). If more than six, designate each gel with an appropriate letter. [084] The separated pre-stained marker bands on each gel are visible to the eye. The pre- stained markers distribute on the gel on the basis of their molecular size during electrophoresis to give a ladder of pre-stained markers on the gel (Table I). Each pre-stained marker has a unique and known molecular size. For example, the 200 kDa marker is found closer to the wells at the top of the gel and the smaller 15 kDa marker is found closer to the bottom of the gel. The position of each marker on the gel indicates the approximate area where proteins of similar molecular sizes have been separated on the gel during electrophoresis (Fig. 2B, enlarged). [085] Using the pre-stained markers as reference points, and a ruler as guides, divide each protein gel into several zones (up to nine zones) by making up to ten horizontal cuts on each gel (Fig. 2B, enlarged). The cuts should be parallel to each other on the same gel. The same number of horizontal cuts should be performed for all gels. Each gel now has nine horizontal strips, representing the nine zones. Starting from top to the bottom of the gel, label the horizontal strips zone 1 to zone 9. For ten protein gels labeled A to J, there should be ten replicates of zone 1 strips. The zone 1 strip from protein gel E is designated as El. Likewise the zone 6 strip of protein gel D is designated as D6.

[086] The next step is to assemble the multi-strip gel for the multi-strip Western blot containing only proteins from a zone of interest. By combining all the strips corresponding to the selected zone of interest from different gels on one filter paper in preparation of electro- transfer, only the proteins in the selected zone will be electro-transferred to a single membrane. Pick Al strip, orientate it so that the pre-stained marker is on the left side, and place the strip on a pre-moist filter paper in a transfer buffer. Pick B 1 strip, orientate the strip the same way, and place next to Al so that Al and Bl would lay side by side and parallel to each other on their long side and the both pre-stained markers are on the left (Fig. 2C). In this arrangement, the strips are in parallel tandem to each other. Repeat the same steps for strips Cl-Fl of gels C to F. [087] After all zone 1 strips are arranged on one filter paper, place a moisten Western blot membrane over the strips to make a sandwich. Place another moist filter paper on the membrane following with sponges and complete building the Western blot transfer sandwich. Place the sandwich into a Western blotting electro-transfer apparatus and proceed with electroblotting. Fig. 3 shows three separate membranes of proteins from zone 4, 5, and 6 respectively separated on ten protein gels A to J.

[088] With the present invention, a single protein gel now can be used to study several proteins by Western blotting compared to the traditional one-protein-gel-one blot-one protein study method. This makes the multi-strip Western blotting procedure very economical, especially for protein samples that are very precious because they are very difficult to obtain and/or are available in very limited quantities. At the same time the invention eliminates the need to strip the membrane to re-blot it with different antibody or to load another set of samples just to quantify some housekeeping proteins that are used as control of sample loading or experimental conditions.

[089] In one embodiment, this invention greatly improves data output by up to 10 fold over a traditional Western blotting technique to quantify protein abundance and protein modification states in the cell. The multi-strip Western blotting procedure permits examination of up to 9 different proteins from one gel lane and transfer up to 100 different or repeated samples on to a single Western blot membrane for 10 X 10 cm, 10 well gels. Larger protein gels with 15 wells will allow assembling of a membrane holding up to 150 samples.

[090] With increased data output, the invention also reduces the amount of data variation resulting from multiple protein gel running conditions and electro-transfer-related errors. The multi-strip Western blotting procedure improves data accuracy by reducing signal error by at least two-fold, allows comparative quantitative analysis of proteins from different gels, produces the data that have smaller statistic variances and therefore is more reliable when comparing to the data obtained by the traditional method.

[091 ] Additionally, the present invention provides the ability to detect picogram amount of proteins. Since the multi-strip procedure involves placing of the same type and size proteins on to the same Western blot membrane, even though the proteins are initially separated in different protein gels, the electro-transfer conditions can be adjusted to ensure the complete as well as even transfer of all proteins of interest for accurate protein quantification. For example, large molecular weigh proteins of > 100 kilo Daltons require more time to electro-transfer to membranes while smaller molecular weight proteins do not. Hence if an assembled multi-strip gel consists of mainly large molecular weight proteins, the scientist can increase the electro- transfer time or add some detergent to optimize transfer efficiency. Conversely, for an assembled multi-strip gel consisting of smaller molecular weight proteins, the scientist can just use the standard electro-transfer conditions of 30 volts for 90 minutes. [092] In one embodiment, the width of the strips can vary, depending on the number and molecular weight of the molecule of interest. For example, a strip can be wider such that it includes proteins within the range of 35-60 kDa. The scientist decides on the proteins of interest, considers the proteins molecular weight according to the Tables 1-9, and then decides on the width of the strip that will allow the proteins of interest be captured within that that strip. [093] In another embodiment, the gel strips containing non-identical zones can be places on to an assembling filter. Fig. 2E presents wider strips (containing two zones) from gels A-C placed in parallel to single zone 6 strips.

[094] In one embodiment, the invention can be used to reduce the interference of non-specific bands on the Western blots. By dividing the protein gel into several zones, the non-specific proteins are usually separated from the protein of interest by virtue of zone separation. A multi- strip gel is assembled using the gel strips of the zones containing the protein of interest. The removal of the non-specific protein zones helps to concentrate the antibodies only on the protein of interest, thus eliminating the non-specific signals and providing more accurate protein quantification data, which reflects the true biological phenomena. The invention also helps to conserve rare and precious limited amounts of antibodies compared to traditional Western blotting analysis. For example, the generation of 100 data points to detect a given protein in a traditional approach will consume 10 volumes more of antibody solution than in the present invention.

[095] The invention allows the quantitative comparative study of many protein samples collected in time-course experiments under various experimental conditions without losing quality of the signals. For example, the expression levels of seven enzymes in a biosynthetic pathway were monitored in the cells that were treated with a given drug over a course of 48 hours, while control cells were left untreated. The samples were taken at every 4 hour intervals, Three independent experiments were conducted. This represents a total of 72 time point samples. Using methods disclosed herein, the 72 sample points can be loaded on six 12-well mini gels and separated by protein electrophoresis. If each enzyme migrates in non-overlapping zone, then each gel can be cut into the seven strips containing these enzymes. A series of three control and three drug-treated cells, one series for each of the 12 time points, are assembled to form a single multi-strip gel. Upon Western blot transfer, a multi-strip blot is produced. A multi-strip blot can be produced for each of the seven enzymes. Thus the whole data set reflecting the expression of each enzyme can be represented as a single membrane after single electroblotting cycle. The traditional Western blotting procedure, in contrast, would produce six separate membranes for each given enzyme. Consequently, the present invention would generate only 7 membranes versus 42 membranes required by the traditional method. Such economical means of data handling allows accurate simultaneous comparison of the enzyme protein expression levels over time under even data-processing conditions

[096] In another embodiment, the present invention provides manual and automated bench- top devices for cutting the protein gel slices into the pre-determined zones, and arranging and assembling the gel slices. These devices meet the needs of researchers to obtain standardized, high throughput quantitative data. The devices enable about a 10-fold more economical, highly productive and accurate side-by-side comparison of proteins' abundance or/and their modification states by ensuring the same electro transfer conditions for about 100-150 different or repeated protein samples compared to 10-15 samples for the traditional method. [097] The devices are designed to uniformly and simultaneously cut a gel into several zonal strips and then align the gel strips for the subsequent multiple-strip Western blotting procedure. [098] The simplest device will serve for manual cutting of commercially available precast mini or midi gels such as those from Invitrogen, herein termed the "Minislicer" or "Midislicer" respectively, for the different sized protein gels they work best with. The Minislicer and Midislicer are designed to cut the gel strips simultaneously but do not align them. The basic design is a cookie cutter cum egg slicer-like idea of simultaneously cutting several strips with one downward motion on to a horizontally placed gel slab.

[099] In one embodiment, the manual slicer comprise a horizontal platform and legs that support the platform horizontally above a horizontal surface, such as a table or a gel. The horizontal platform can be square or rectangle, and there is a rectangular hollow in the center of the platform. The size of this hollow is at least 10% larger that a gel the slicer is designed to cut. For example, if the slicer cuts mini protein gels of 8.6 X 6.8 cm, the hollow should have dimensions of at least 9.46 X 7.48 cm. Within the hollow center rectangle are several cutting knives mounted across the long width of the hollow rectangle. The knives are mounted such that their sharp cutting surface faces downwards and they are parallel to each other. The knives' positions relative to each other and in the hollow rectangle are adjustable. [0100] In one embodiment, the manual slicer is equipped with means of holding a plurality of knives within the hollow rectangle in the center.

[0101 ] In one embodiment, the height of legs of the platform are adjustable and lockable. In one embodiment, the height of legs can be reduced when a downward force is applied on the platform. In another embodiment, after the downward force have been removed, the height of legs can re-adjust back to the starting original height before experiencing a downward force. In yet another embodiment, the legs are equipped with means of locking the height of the legs, such that the height of the legs do not change upon application of a downward force. [0102] An example of a manual slicer is presented. The Minislicer device (Fig. 10A) consists of, for example, a single 14 x 14 cm platform frame on four legs. As an example, the frame is a little bit larger (~ 2 cm) than a standard 10x10 cm (gel cassette) mini gel. The platform frame can be transparent. For example, there is a 6 X 8 cm hollowed rectangle in the center of the frame. In one embodiment, horizontally parallel blades measuring the length of the hollowed rectangle are mounted on this center hollowed rectangle. For example, the blades are about 8 cm for the 6 X 8 hollowed rectangle in a larger platform frame. For example, the larger platform frame can be 14 X 14 cm. The cutting surface of the blades face downwards. The blade-filled center hollowed rectangle should correspond to the size of the protein gel it will be cutting into gel strips. The positions of the blades are in fixed position or can be adjustable to obtain different sized slices from one gel. For example, in one embodiment, each side of a knife has a hollow prong with a thread, which is inserted into the channel and held in a desired position by a screw-bolt fixer with a screw-cap that is larger than the width of the channel (Fig. 10C). The channel is a cavity where all fixers can move along the frame-impregnated millimeter-scale ruler, thus bringing the knives into adjustable position. The position of each knife to cut a gel strip can be selected from, for example, Tables 2-9, which can be supplied together with the device. Table values can also be found empirically. The present inventors have created a comprehensive database of various molecular weight protein migrations, which determines the exact gel cutting positions The database is based on experimental measurements of pre-stained molecular weight marker migration in different types of precast gels (e.g. polyacrylamide percentage content, buffer system and gel size) that are available on the market. In use, the first knife is aligned with a dye line in the bottom of the gel. If the maximal number of gel strips is not required, unnecessary knives may be easily removed out of the frame. [0103] In one embodiment, the platform frame can have legs with springs at the four corners of the frame (Fig. 10). The spring of the leg has a locking mechanism to prevent accidental downward push and cutting of gel before the platform is correctly positioned. Due to the springs within each leg of the device, the gap between gel and knives is maintained (UP in Fig. 10B). After all knives' positions are fixed within both channels by tightly screwing the fixers (Fig. 10C), the user simply pushes down the device in order to cut the gel into the desired slices (DOWN in Fig. 10B). Once the position of knives is fixed, the series of gels can be sliced into the identical strips in a very short time with the following manual combination of appropriate slices on to the same membrane.

[0104] To use the device, one places the whole platform horizontally over a slab gel so that the blades are above the gel and cover the gel completely, the legs are unlocked, and the device is pushed uniformly downwards to cut gel. Upon released, the platform and blades will spring upwards.

[0105] In another embodiment, the device is semi-automated combined multi-gel slicer and slice assembling processor that is controlled by an interactive software with user-friendly interface for the selection of proteins to be cut out of the gel and analyzed, termed herein the "AutoSlicer". It is capable of cutting and processing of up to 10 gels simultaneously by using laser beams (Beam AutoSlicer) or sharp knives (Blade AutoSlicer). In one embodiment, the device consists of two-level compartments and touch-screen for point-and-click directions. The drop-down menu in a touch-screen allows user to choose the number and the type (percentage and brand) of ready-to-be-processed gels following with the selection of protein entries under the investigation

[0106] One part of the device does the gel slicing (level 1) and can handle up to 10 gels at one time (Fig. 11). The gel slices are supported by a filter beneath the gel. The other part of the apparatus is the assembler (level 2) (Fig. 11). The first level has, for example, ten chambers for placing the filters with full sized gels immediately after gel electrophoresis and a set of sharp knives or the source of laser beam above. A program asks to choose up to 10 signaling proteins that user wants to visualize from each gel from pre-loaded list. If the protein is not listed, but user knows the exact position of its migration from the dye line in a certain type of the gel, he may be able to manually add the protein in the list, such as those in Table 1-9. Similarly, the user can modify the pre-loaded database, for example, reduce or increase cutting area for a certain protein and save the changes in a specified directory. If the cutting areas interfere with each other, the program warns the user about the incompatibility. After the final confirmation, the device automatically directs laser beam or puts appropriate cutting knives in positions to slice the gel into the strips with user-defined proteins inside. Respective positions are determined by installed color-sensitive detector, which tracks the final location of the dye-line and a processor, which sets an algorithm for mechanical positioning of the blades up from the dye-line with reference to a given database. After this procedure, the given strip from each gel together with an underlying filter is transferred to the second-level compartment, where the propulsive piston moves it on to the assembling area.

[0107] The assembling area includes, for example, a tray with sponge-pads and additional filter paper. After one slice has been placed on the assembler, a wide propulsive piston arm spanning the length of the gel slice pushes the gel slice to the far end of the assembler on to a bed of sponge and filters. The propulsive piston arm is retracted and a new gel slice is transferred on to the assembler. The propulsive piston arm again pushes the second gel slice to the far end, up next to the first gel slice, stops and retracts itself. The cycle repeats till gel slices completely cover the bed of sponge and filter at the far end of the assembler (Fig. 11). The entire assembly of sponge filter and gel slices can now be removed out of the tray and placed directly into the external blotting module of choice, covered with a Western blot membrane (nitrocellulose, PVDF, nylon membrane or others) and other materials required to perform the standard electro transfer (Fig. 11).

[0108] In one embodiment, the AutoSlicer is suited for integrating into the automated separation of electrophoretic analytes by horizontal SDS electrophoresis (H-SDS) (US Patent No. 5338426). Principles of horizontal gel casting procedure is well-described by Serva company. Serva, Isogen Life Science and other companies already offers ready-to-use precast gels for protein separation by H-SDS. The user supplies the dispenser unit of device with different samples in the order they must be loaded on to the gel. The samples are precisely applied on to gel wells by integrated and highly accurate applicator. H-SDS electrophoresis is simultaneously performed in every chamber under the same conditions until the dye-line reaches the bottom of each gel. This step is monitored by color-sensitive detector. Once electrophoresis process is stopped, the following step of gel cutting takes place.

[0109] In another embodiment, the invention provides for the PreCut'n'Cast gels that can be used to separate proteins by conventional SDS electrophoresis (Fig. 12). PreCut'n'Cast gels are precast gels that are already divided into several pre-determined gel strips corresponding to several zones, hence the term "PreCut". No cutting of the gel is required after the biological sample have been separated. The pre-determined strips of interest can be simply separated from the adjacent strips that make up the PreCut'n'Cast gel and be used to assemble a multi-strip gel for Western blotting. The usage of such gels eliminates the need of any devices described above. The gels are horizontally casted by a manufacturer in precut-form of multiple strips and assembled into the standard gel-cassette, which is later used for sample loading and protein separation. Gel materials are initially casted horizontally to produce multiple strips of various width. These strips are then assembled to form a standard gel cassette. In one embodiment, the width of the precut strips in a PreCut'n'Cast gel is uniform, such as shown on Fig. 12 A. This gel is designed to capture proteins of various molecular sizes in the middle of desired strips by varying the running time of electrophoresis. In another embodiment, the width of the precut strips in a PreCut'n'Cast gel is non-uniform and covers a range, such as shown in Fig. 12B and C. Such PreCut'n'Cast gels are customized for capturing specific signaling proteins of specific molecular weights according to Tables 1-9.

[01 10] In one embodiment, the PreCut'n'Cast gels include polyacrylamide gels and agarose gels.

[01 1 1 ] In one embodiment, the PreCut'n'Cast gel can be made as a single piece of gel casted in the gel cassette. Upon solidification, the gel cassette is opened to gain access to the gel. The single piece of gel is then cut into the strips using a sharp blade in horizontal or vertical setting. The gel cassette is closed afterwards. In one embodiment, the cutting procedure does not affect the migration of proteins in the gel strips within the assembled gels, as presented in Fig.16. This method of making PreCuf'n'Cast gels produces only single percentage gels. [01 12] PreCut'n'Cast gels can also be made so as to include the gel preparation of both fixed (e.g. containing 8%, 10% or 12% polyacrylamide etc.) and gradient (e.g. from 4 to 12% polyacrylamide) percentages.

[01 13] In one embodiment, small portions of gel of a specific gel percentage are casted sequentially in a vertical or horizontal cassette. The first portion is allowed to solidify before the addition of the next portion of gel. A small amount of low density gel polyacrylamide or agarose is added on top of the solidified first portion prior to adding the second portion. This process is repeated until a complete gel is casted in the gel cassette. The solidified gel portion forms a gel strip. In a polyacrylamide gel, each strip is separated from the neighboring strip by a narrow region of a low density polyacrylamide. Specifically, starting from the bottom, the sufficient volume of liquid polyacrylamide (e.g., 5 ml) of fixed density and greater than 4% (e.g., 8%, 10% or more) is poured into the cassette for the first (bottom) strip. Polyacrylamide solution contains gel strengthener additive (e.g. Rhinohide, Invitrogen), which makes gel much stronger. After it solidifies a small amount of lower density polyacrylamide (e.g., 3 or 4%; without strengthener), which is able to solidify, is poured to form a narrow (1-2 mm) boundary, separating two neighboring slices. The procedure is repeated to form the next gel strip until as many as nine strips for different molecular weight proteins are formed (Fig. 12). [01 14] In another embodiment, the gel slices are prepared in a similar manner to as described above, but they are separated by a thin strip of plastic film. Such film is solid but porous and transparent for protein migration in the electrical field. Thereby the film allows separating the neighboring gel slices after polyacrylamide gel electrophoresis (PAGE) under native or denaturing conditions. Moreover, these films can be exploited to separate different slices following the opening of the gel cassette if one side of the film surface is treated in order to attach to the polyacrylamide gel, and another side is not. Such films are readily available (e.g. polyester NetFix film from Serva).

[01 15] This invention is further illustrated by the following examples which should not be construed as limiting. The contents of all references, patents, and patent applications cited throughout this application, as well as the figures and tables are incorporated herein by reference. EXAMPLE 1 Applications of the multi-strip Western blotting procedure. [01 16] Here the inventors consider the major sources of error in immunoblotting and highlight important factors that limit the efficiency of quantitative analysis. The inventors propose a simple modification of the Western blotting procedure that increases throughput without loss of sensitivity and enables reliable side-by-side comparisons of multiple samples thus saving time, immunoblotting reagents and costly antibodies. [01 17] Materials and methods [01 18] Cells, reagents and antibodies

[01 19] Human epithelial lung carcinoma (A549), bladder carcinoma (T24), embryonic kidney (HEK293) and mammary epithelial (MCF-IOA) cells (ATCC, Manassas, VA) were maintained in Ham's F-12, McCoy's 5A, DMEM/F-12 (all from GIBCO, Grand Island, NY) and mammary epithelium basal medium (Cambrex Biosceinces, Walkersville, MD), respectively, supplemented with 10% fetal bovine serum (GIBCO, Grand Island, NY) and penicillin- streptomycin solution (100 μg/ml each) (Mediatech, Herndon, VA) in a humidified 5% CO₂ incubator at 37⁰C. Routine chemical reagents were obtained from Fisher Scientific (Pittsburgh, PA), unless otherwise noted. Heat inactivated BSA was purchased from Roche Diagnostics (Indianapolis, IN). Human recombinant epidermal growth factor (EGF), hepatocyte growth factor (HGF) and insulin-like growth factor 1 (IGF-I) was from PeproTech (Rocky Hill, NJ). Recombinant active MAPK was provided by Upstate (Charlottesville, VA). Wortmannin (Cell Signaling, Beverley, MA) was dissolved in dimethyl sulfoxide (DMSO) to 1 mM stock concentration and diluted to final concentration of 100 nM in cell culture medium. Bisindolylmaleimide I hydrochloride was obtained from Calbiochem (San Diego, CA). The following antibodies were used: polyclonal anti-phospho-SHP2 (Tyr542) (all from Cell Signaling, Beverley, MA), anti-GRB2 (C-23), anti-SHP2 (C-18), anti-GABl (H-198), anti- phospho-GABl (Tyr627) (all from Santa Cruz, Santa Cruz, CA), anti-phospho-EGFR (Tyrl 173) (BioSource, Camarillo, CA), anti-PI3K-p85 (Upstate, Charlottesville, VA), monoclonal anti- phospho-MAPK (Thr202/Tyr204), anti-phospho-AKT (Ser473) (587Fl 1) (both from Cell Signaling, Beverley, MA), anti-RasGAP (B4F8) and anti-α-Tubulin (DMlA) (both from Upstate, Charlottesville, VA). Prestained Precision Plus Protein standards marker was from BIO-RAD Laboratories (Hercules, CA). Blotting filter paper (2.5 mm, 7x10 cm) was purchased from E&K Scientific (Santa Clara, CA). [0120] Cell stimulation, lysis and sample preparation

[0121 ] Routinely 2xlO⁶ cells were seeded in 60 x 15 mm tissue culture plates (Fisher Scientific, Pittsburgh, PA) and cultured for 48 hours before lysis. For EGF-response studies, the cells were starved for additional 16 hours in serum-free medium and stimulated with a range of EGF concentrations for different times. For inhibitor studies the cells were pretreated with 100 nM of wortmannin for 30 minutes or with 1 μM of Bisindolylmaleimide I for 1 hour prior to stimulation. Control cells were treated with DMSO. After stimulation, the culture medium with EGF was removed, and cells were lysed in ice-cold lysis buffer (150 mM NaCl, 2.5 tnM EGTA (pH 7.4), 25 mM HEPES (pH 7.4), 5% glycerol, 1% Igepal CA-630, 1% Triton X-100 (both from Sigma, St Louis, MO) containing 10 μl/ml Phosphatase Inhibitor Cocktail Set II (Calbiochem, San Diego, CA) and one Complete protease inhibitor cocktail tablet per 10 ml (Roche Diagnostics, Indianapolis, IN). Detergent-insoluble material was removed by centrifugation at 10,000 x g for 10 min at 4^°C. Protein concentrations were determined using the bicinchoninic acid solution assay (Pierce Biotechnology, Rockford, IL). For SDS-PAGE, an equal amount of total protein was dissolved in 5x Laemmli buffer (BIO-RAD Laboratories, Hercules, CA) with 5% β-mercaptoethanol and boiled for 5 min at 95^°C. For LDS-PAGE (NuP AGE® in Invitrogen, Carlsbad, CA), fraction with solubilised proteins was dissolved in 4x NuPAGE LDS Sample Buffer supplemented with NuPAGE Sample Reducing Agent (5OmM DTT) (both from Invitrogen, Carlsbad, CA) and heated for 5 min at 75^°C. Elecrophoresis and Immunoblotting

[0122] An aliquot of 10 μl of Prestained Precision Plus Protein standards was loaded in one lane. Equal volumes of prepared lysates were subjected to LDS-PAGE using Novex 4-12% gradient Bis-Tris gels for protein separation under reducing conditions in MOPS-SDS running buffer (50 mM MOPS, 5OmM Tris Base, 0.1% (w/v) SDS, ImM EDTA, pH 7.7) supplemented with NuPAGE Antioxidant (all materials from Invitrogen, Carlsbad, CA) at 150V. SDS-PAGE was performed using Novex 8% Tris-Glycine gels in Tris-Glycine-SDS running buffer (25 mM Tris Base, 192 mM Glycine, 0.05% (w/v) SDS, pH 8.3) at 100V. Resolved proteins were transferred on to 0.2 μm nitrocellulose membrane (BIO-RAD Laboratories, Hercules, CA) in XCeIl II Blot Module (Invitrogen, Carlsbad, CA) for 1.5 hours at 30V constant using NuPAGE transfer buffer (Invitrogen, Carlsbad, CA). The membranes were blocked with 4% BSA in Tris- buffered saline (10 mM Tris-HCl (pH 8.0), 150 mM NaCl) plus 0.5% (w/v) Triton X-100 (TBS- T buffer) for 2 hours at room temperature, then incubated with indicated primary antibodies overnight at 4^°C. After extensive washing with TBS-T, the membranes were incubated with a 1:50,000 dilution of HRP-conjugated goat anti-rabbit IgG (Pierce Biotechnology, Rockford, IL) or horse anti-mouse IgG (Cell Signaling, Beverley, MA) at 1: 10,000 dilution for 1 hour at room temperature and washed with TBS-T again. Data evaluation [0123] The immunoreactive bands were detected by KODAK Image Station 440CF after treating membranes with a working solution of the SuperSignal West Dura Extended Duration Substrate (Pierce Biotechnology, Rockford, BL) for 5 minutes. Quantitative analysis of bands was performed by KODAK Digital Science software (Kodak Scientific Imaging Systems, New Haven, CT). To enable comparison, the capture time and number of frames was equal for each separately exposed membrane or the whole membranes were exposed simultaneously. Results were plotted and statistically analyzed using Microsoft Excel program. [0124] Results

[0125] Gel-related band artifacts that impair quality of the signal.

[0126] A wide variety of precast gels is commercially available that vary in gel composition, percentage, thickness, number of well and buffer systems. The major disadvantage of commercially obtained gels is variability in batch-to-batch quality. Despite these drawbacks, precast gels save time and have lower gel-to-gel variations than self-prepared gels. The proteins resolved by high quality gels in freshly prepared buffer should result in crisp and straight bands that are not distorted (Fig. 8). The older a gel, the higher the number of poor quality bands was detected in our experiments. Old Bis-Tris or Tris-Glycine midi-gels gave mostly winged and wavy bands; old Tris-Glycine mini-gels usually provided faded, fuzzy and ribbon-like bands, while faint, somewhat faded, winged or smiley bands were associated with old Bis-Tris mini- gels (Fig. 8). Here the 105 kDa GABl protein was detected in HEK293 cell lysates, resolved by 8% Tris-Glycine and 4-12% gradient Bis-Tris 10- well gels used two months before and after their expiration date. In general, 4-12% gradient NuPAGE mini-gels, which are fairly homogenous, have long shelf life, demonstrated excellent protein separation and were suitable to generate better quality bands in comparison with regular Tris-Glycine gels even after their expiration date (Fig. 8B).

[0127] There are many other factors that influence band quality (Fig. 8A). For example, an excess of SDS in transfer buffer and lack of contact between gel and the membrane during transfer may result in bubbly and dotted bands. Wavy, faint or diffuse bands may occur, when insufficient protein is loaded, protein binding to the membrane is weak, there is a variation in pressure between the gel and the membrane during transfer, or the time of transfer for certain molecular weight protein is not optimized. The use of fresh and high quality SDS in sample preparation markedly increases the sharpness of bands. Ribbon-like, dotted, sliced and streaked bands may form as a result of poor transfer conditions but typically require checking concentration and potency of primary antibodies. Streaked bands may also occur due to poor separating gel quality, leading to non-uniform protein concentration across the lane or high sample viscosity. Non-uniform heat distribution through the gel matrix and slightly slower migration of first and last lanes during electrophoresis due to inhomogeneous electric field may cause smiley bands, occurring at the base and at the edges of the gel. Overheated gel looses rigidity, leading to poor resolving and blurry bands. Incorrect sample buffer-to-protein ratio, overheating during preparation step, failure to remove insoluble material, overloading or under loading protein sample may also cause band artifacts.

[0128] Although the visualization of most bands is adequate to confirm "yes-or-no" protein expression or compare "on-off ' cell responses to a stimulus, only intense bands with well- delineated borders should be used for quantitative analysis. [0129] Quality versus quantity problem

[0130] The intensity of chemiluminescence emission is linearly proportional to the protein concentration over a broad range. However, the lack of linearity in the low- and high-ends of the signal makes these areas unsuitable for quantitative measurements of protein abundance. Light quenching, rapid substrate consumption and enzyme inactivation by oxidation reaction- generated free radicals leads to the saturation of a strong signal, while non-linearity of the low- end signal is due to sensitivity limitations of reaction rate accelerator (enhancer) and delayed enzyme kinetics. Dilution of a sample can shift a strong signal to the linear range, but when the protein concentration in the sample is low, the use of more sensitive substrate (e.g. SuperSignal West Femto Maximum Sensitivity Substrate from Pierce) may increase low-end linearity and broaden the lower limit of detectability. Additionally, the loading volume per lane must be substantially increased to gain a stronger signal. If a large number of samples need to be compared, the number of lanes per gel becomes a limiting factor raising the "quality versus quantity" dilemma.

[0131 ] Several manufacturers (Invitrogen, Cambrex, LifeGels) offer precast mini-gels for vertical protein electrophoresis, enabling the loading of a larger number of samples per gel (e.g. 15, 17 well), resulting in apparently increased throughput. Unfortunately, thin gels with many small lanes are restricted to lower sample loading volume and consequently the sensitivity of the signal is sacrificed in order to gain higher throughput. In response to this problem the manufacturers started to offer larger and thicker midi-gels (e.g. 18, 20, 26 wells), which can accommodate a larger sample volume per lane (Invitrogen, BIO-RAD). However, the size of midi-gels dramatically augments errors caused by increased gel heterogeneity combined with unequal electrophoresis and protein transfer conditions (Fig. 1).

[0132] In Fig. 1, A549 cells were cultured and lysed and ten unique samples were prepared (indicated as digits). Equal volume of the same lysate was loaded in two non-neighboring wells of Novex 4-12% gradient Bis-Tris 20-well midi-gels. Proteins were resolved by LDS-PAGE under reducing conditions and independently transferred on to nitrocellulose membranes in Criterion Cell Blotter (30V constant for 1.5 hours). The blocked membranes were blotted with polyclonal antibodies that recognize 28 kDa GRB2 at a dilution 1:2,000 with successive procedures described in "Materials and Methods" and collectively exposed. A representative experiment is shown, but similar results were observed in seven separate experiments, including three performed with Novex 14% Tris-Glycine 15-well gels.

[0133] Non-uniform heat distribution through the long gel matrix and slightly slower migration of first and last lanes during electrophoresis due to inhomogeneous electric field caused dissimilar signal intensities from identical samples that were randomly loaded in different lanes. The optimal protein transfer region covers approximately ten neighboring lanes in the center and is less adequate on the edges of the midi-gel.

[0134] A very helpful strategy of randomized loading was proposed to reduce correlated errors caused by gel in homogeneity [46]. However, this approach does not eliminate the major source of errors, i.e. electrophoresis and immunoblotting procedures that involve many factors: sampling variance [47], quality and age of the gel (Fig.8), composition of buffer systems [39, 40, 48-50], type of blot module, materials for electroblotting (e.g. firmness of filter paper, shape of fiber pads, type of the membrane support (nitrocellulose or PVDF) [51, 52]), optimized time of protein transfer on to the membrane [35, 39], quality and dilution of antibodies to optimize signal-to-noise ratio, membrane washing [36], signal detection [41] and normalization of raw data [53].

[0135] Multi-strip Western blotting procedure

[0136] The presence of numerous sources of errors makes it difficult to produce a large number of high quality quantifiable data points. The simple and practical procedure, which would improve immunoblotting accuracy, maintaining high sensitivity, is necessary to solve "quantity versus quality" problem.

[0137] Here the inventors developed a novel modification of Western blotting procedure, which, compared to the previously mentioned system, maintains high sensitivity with limits of detection of 1000 to 2500 fold smaller amounts of protein, does not require any additional tools and functions using any conventional gel electrophoresis systems. Although the following modification protocol is described for Novex 4-12% gradient NuPAGE mini-gels and prestained Precision Plus Protein marker (see Materials and Methods), it can be applied to any type of preferred mini- or midi-size gels and prestained protein size markers. [0138] Several sets of samples are loaded on to six gels (Fig. 2A - gels A to F) for electrophoresis (up to ten gels may be used). Protein molecular weight marker is loaded on to the first and/or the last lane of each gel. Separation should be started with an interval of 5-10 minutes between tandem electrophoresis units to reserve enough time for follow-up steps. The measurement of protein molecular weight marker (M) migration relative to the dye line (H) and identification of protein migration zones are shown as numbers in circle. Representative protein within each migration zone is depicted as a set of horizontal bands (dashed line). Scissor symbols indicate the position of the cutting lines, which would separate the entire gel into several strips.

[0139] When a dye line reaches the bottom of gel A, protein separation is stopped and the gel cassette is gently opened with a gel knife. The gel should remain on one of the plates. Then a centimeter-scaled transparent ruler is firmly positioned near the edge lane with a separated marker, so that zero (0 cm) aligns with the dye line and the distance from the dye line to the center of each marker band (H) is measured in millimeters (Fig. 2B). The distance between two marker bands corresponds to the migration range of certain molecular weight proteins. For example, the distance between H₂₅₀ and H₁₅0 defines a migration range of various proteins with molecular size between 150 and 250 kDa and is shown as zone 1 in Fig. 2B. The strip, which covers an area with a protein of interest located in the middle, can be precisely cut out from the gel using the gel knife. Each unique sample may provide up to the nine protein-containing areas that may be simultaneously cut out from the same gel. The number of strips to be cut out from one gel depends on the number of distinct proteins to be detected. The most frequently studied signal transduction proteins of various migration zones are listed in Table 1, which also indicates the appropriate areas to be cut out of the gel. For example, phosphorylated AKT has a molecular weight of 60 kDa and may be readily separated from activated 95 kDa insulin receptor and both 42 kDa ERKl and 44 kDa ERK2 isoforms by cutting the gel into three strips at 22, 29, 38 and 44 mm from the dye line (Table 1). The gel outside the strips is discarded, and the plate with remaining strips is covered with moistened filter paper. Similarly, identical strips from other gels (B through F) are prepared.

[0140] To avoid confusion, we assigned a code for each strip: gel C zone 5 indicates that the strip was derived from gel C and contains proteins with molecular sizes between 37 and 50 kDa; gel D zone 4+5 - was cut out from the gel D and contains proteins with molecular sizes between 75 and 37 kDa, etc. If a protein of interest lies in the intersection of marker-defined zones (e.g. 100 kDa GABl protein, which migrates between zones 2 and 3), the cutting area can be wider. [0141 ] The strips are assembled on to the membrane in different combinations for the subsequent electroblotting. In the next step, the strips derived from different gels are combined on to the same assembling filter paper. The maximal number of strips that can be placed on to one assembling filter depends on its size (height and width). Routinely we use 2.5 mm thick 7x10 cm filter paper, which provides space for twelve gel strips of 0.6 cm height each. The plate with gel A strips is flipped and gently lifted so that all strips would stick to the moistened filter paper. A gel knife can be used if the strips do not independently detach from the plate. After the first strip (e.g. containing zone ® proteins) is transferred on to the assembling filter paper, the filter with remaining strips is returned on to the plate by flipping it back. Similarly, the first strip from the plate with gel B, gel C and other plates is sequentially transferred on to the assembling filter. When all zone 1 strips are combined on to the assembling filter paper, it is placed on the fiber pads in the transfer unit, covered with a piece of nitrocellulose membrane, other moistened filters and fiber pads. Fig. 2C shows the schematic picture of the resulting blot after transfer and immunoblotting for protein of interest. The second strip (e.g. containing zone 2+3 proteins) is similarly assembled and transferred on to the appropriate membrane (Fig. 2D). The same procedure is performed with the remaining strips.

[0142] There are, of course, other possible combinations of laying strips on to the assembling filter. Fig. 2E shows the bands of two different zone proteins transferred on to the same membrane. This situation is especially favored for the detection of phosphorylated/non- phosphorylated and housekeeping proteins (e.g. tyrosine phosphorylated 66, 52 and 46 kDa SHC isoforms, which co-migrate in zone 4+5, normalized by total protein level of 28 kDa GRB2 from zone 6). Similarly, Fig. 2F illustrates the bands of three different zone proteins transferred on to the same membrane (e.g. serine-phosphorylated 60 kDa AKT from zone 4 and serine- phosphorylated 45 kDa MEK1/2 from zone 5 both normalized by total protein level of 37 kDa GAPDH from zone 6). In these cases, the blocked membrane can be incubated with the mixture of primary antibodies or can be cut into pieces and incubated with appropriate primary antibodies in separate dishes. Such membrane pieces may be imaged all together (e.g. phospho- AKT, phospho-MEK and GAPDH), in combination (e.g. the first piece: phospho-AKT and phospho-MEK, the second piece: GAPDH) or separately. [0143] Increased data throughput using multi-strip Western blotting procedure [0144] Fig. 3 shows A549 cells that were cultured, starved, left untreated or stimulated with 1 nM EGF for 5 minutes, and lysed. Equal volumes of non-stimulated-cell lysate was loaded on to odd wells (1, 3, 5, 7, 9), while stimulated-cell lysate was loaded on to even lanes (2, 4, 6, 8, 10) of ten (A through J) Novex 4-12% gradient Bis-Tris 10-welI mini-gels. Proteins were resolved by LDS-PAGE under reducing conditions. Each gel was divided into six strips containing proteins from zone 1 to 7, according to the Precision Plus Protein standards marker migration statistics from Table 1. The same protein zone strips from gels A to J (indicated by hand symbols) were combined on to appropriate assembling filter papers. The proteins from each assembling filter were respectively transferred on to nitrocellulose membrane and immunoblotted for various proteins. Representative blots with ten replicates of phosphorylated (p) p-SHP2 (Tyr542) (upper panel - zone 4) (A); p-ERK (middle panel - zone 5) (B); and GRB2 (bottom panel - zone 6) (C) are shown. Other detected proteins were p-EGFR (Tyrl 173) (zone 1), p-GABl (Tyr627) (zone 2+3) and Ras (zone 7) (not shown).

[0145] The suggested multi-strip procedure is very economical: several different proteins may be analyzed from one load of precious sample in one immunoblotting cycle. Furthermore, our modification permits examining up to ten different proteins from one gel lane and transfer up to one hundred different or repeated samples on to a single membrane (Fig. 3), in addition dramatically saving costly antibodies. If gel systems with more wells were used, the data output would increase proportionally. For example, a multi-strip procedure using 15-well gels would generate a membrane holding up to 150 bands, originated from either unique or replicate samples.

[0146] In addition, the inventors show increased reproducibility of both high and low signals in multi-strip Western blotting. In Fig. 4A, two different dilutions (100% and 50%) of HEK293 cell lysate were prepared and loaded on to three (A, B, C) Novex 4-12% gradient Bis-Tris 10- well gels. Equal volumes of 100% lysate was loaded on to odd wells of gels A and C (lanes 1, 3, 5, 7) and on to even wells of gel B (lanes 2, 4, 6, 8). 50% lysate was loaded on to the rest of the wells. Proteins were separated by LDS-PAGE. Zone 2 strip with proteins of molecular weight from 100 kDa to 150 kDa was cut from each gel according to the migration of 10 μl Precision Plus Protein standards marker (M). All three strips were combined on one assembling filter paper. The proteins were transferred on to the same nitrocellulose membrane and immunoblotted (IB) for RasGAP protein. The signals from the multi-strip Western blot appear are very reproducible from different protein gels and correlates with the protein load. [0147] In Fig. 4B, starved HEK293 cells were stimulated with 1 nM EGF for indicated time periods and lysed. Identical sample sets were loaded on to four Novex 4-12% gradient Bis-Tris 10-well gels (A-D) for LDS-PAGE. Zone 4 strips were cut out, according to the migration of 10 μl Precision Plus Protein standards marker statistics from Table 1, combined on to assembling filter paper, covered with nitrocellulose membrane and electroblotted. The BSA-blocked membrane was cut into two pieces. The first piece was immunoblotted (IB) for p-SHP2, the second - for SHP2. The signals are very reproducible from different protein gels and correlates with the protein load at low target protein.

[0148] Statistical analysis of data is required to differentiate between functionally significant effects and differences caused by sampling variation. For reliability, at least three independent experiments should be performed, where all experimental data points must be run at least in triplicate. Inhomogeneity of the gel, which generally depends on its age and size, can severely impair the quality of replicates, increasing data variability. The use of smaller fresh gels reduces errors caused by gel inhomogeneity, while multi-strip transfer dramatically increases the number of simultaneously detectable bands. Therefore the inventors approach is suitable for generating multiplicates with highly reproducible signal, which does not depend on the lane position within the gel (Fig. 4A) or amplitude of the signal (Fig. 4B). [0149] Multi-strip Western blotting reduces transfer-derived error

[0150] The inventors tested if protein transfer on to the same membrane under equal transfer conditions (the same pH, methanol/Tris-Gly/SDS concentration, transfer time, strength of the contact between gel and the membrane etc.) attenuates transfer-based signal variability (Fig. 5). To compare the standard deviations of signal intensities, decreasing concentrations of recombinant protein (Fig. 5A) or proteins of various molecular weights from fixed-concentration samples (Fig. 5B) were transferred i) on to three separate gel-sized membranes or ii) a single membrane by means of multi-strip modified procedure.

[0151 ] In Fig. 5 A, serial dilutions (1: 1.5) of active recombinant MAP kinase protein (aERK) were used to generate the calibration curve derived from immunoblotting of protein samples either on separate membranes ("Separate transfer" - solid line) or on one membrane ("Multi- strip transfer" - dashed line). Upper panel shows the whole linear and non-linear dynamic range of the chemiluminescent signal. Zoom of low-end signal is displayed in the lower panel. Error bars represent the standard deviation of the mean of triplicate measurements. [0152] In Fig. 5B, MCF-IOA cells were cultured, starved, stimulated with 10 nM EGF for 10 minutes, and lysates were prepared as described under section "Materials and Methods". Eight replicates of the same sample were loaded on each of the six No vex 4-12% gradient Bis-Tris 10- well mini-gels (gel 1 through gel 6). Proteins were separated by LDS-PAGE under reducing conditions. Gels 1, 2 and 3 were covered with nitrocellulose membranes and subjected to protein transfer in separate blot modules. The membranes were cut into six pieces that were separately immunoblotted for phospho-EGFR, phospho-GAB 1 , PDK-p85, phospho-SHP2, phospho- ERK 1/2 and GRB2 proteins. Each of these proteins was detected by exposing the appropriate membrane pieces from gels 1, 2, 3 simultaneously ("Separate transfer" bars). Alternatively, gels 4, 5 and 6 were used in multi-strip blotting procedure. For detection of each selected protein listed above, three corresponding gel strips were subjected to transfer on to single membrane ("Multi-strip transfer" bars) and immunoblotted. Bars represent the mean of signal intensities of twenty four bands (eight sample replicates from three gels) with error bars corresponding to standard deviation of the mean.

[0153] The results indicate that the relationship between the chemiluminescence signal and the quantity of recombinant protein is sigmoidal. The detection limit for recombinant protein was 150 pg per lane in our system. The signal became non-linear above 35 ng of protein per lane. The variability was more pronounced at low rather than high signal intensity, and multi-strip procedure was especially favorable for the improvement of faint signal quality from 2.5 to 4.3 fold (Fig. 5A).

[0154] The large surface of gel-sized membranes is often unequally covered with immunoblotting reagents, increasing uneven background. When whole membrane is immunoblotted for a single protein, poor antibodies may additionally cause the occurrence of non-specific bands that interfere or overshadow weaker specific signals (Fig. 9). The nonspecific protein bands can be bypassed by narrowing detection area. HEK293 cells were transfected with GAB 1 -specific small interfering RNA (siRNA) for 72 hours as described previously [29]. The cell lysates were prepared, separated by SDS-PAGE and immunoblotted for total GABl protein. Arrow sign shows 105 kDa GABl protein, which was specifically suppressed by siRNA. Scissors in the left panel indicates the suggested cutting area of the gel in order to eliminate the detection of non-specific bands (indicated by asterisk symbol). The improved signal after elimination of non-specific bands is shown in the right panel. Cutting a gel strip with a protein of interest in the middle helps to concentrate antibodies on the more narrow area of the membrane in subsequent incubation steps after protein transfer, and to diminish these drawbacks.

[0155] Application of multi-strip Western blotting for comparative analysis [0156] The best comparative quantitative analysis could be achieved, when the samples are loaded on to the same gel, because each electrophoretic and following immunoblotting cycles are performed under slightly different conditions (which increases data variability and thus restricts comparative quantitative analysis of bands that are visualized on different blots). But when the number of samples exceeds the number of lanes of one gel and multiple data points from one or several experiments should be plotted and compared in a single graph, our modified Western blotting procedure becomes a necessity. [0157] Fig. 6A illustrates an experiment that was designed to compare the activation kinetics of phosphorylated ERK protein from zone 5 (left panel) under three different stimulation conditions and in the presence of one perturbation. The cells were treated with increasing concentrations of EGF for various periods of time in the presence or absence of the general PKC inhibitor Bisindolylmaleimide I. HEK293 cells were starved overnight, and prior to stimulation incubated with 1 μM of Bisindolylmaleimide I or DMSO for 1 hour. Inhibitor untreated cells were incubated with DMSO (gel A), 0.1 nM (gel E), 1 nM (gel F) or 10 nM (gel G) of EGF for 1.5, 3, 5, 7.5, 10, 15, 30 minutes or left unstimulated (0 min). Inhibitor-treated cells were stimulated with 0.1 nM (gel B), 1 nM (gel C) or 10 nM (gel D) of EGF for the same periods of time of left unstimulated (0 min). Equal volumes of appropriate set of samples were loaded on to Novex 4-12% gradient Bis-Tris 10-well mini-gels A through G, and separated by LDS-PAGE under reducing conditions. Zone 5 (right panel), zone 6 (left panel) and other zone (not shown) strips were cut out of each gel, according to the migration of 10 μl Precision Plus Protein standards marker (M). The same zone strips from each gel were combined on to assembling filter paper, covered with nitrocellulose membrane and electroblotted. The membrane with transferred zone proteins was immunoblotted for phospho-ERK (left panel). The membrane with zone proteins was immunoblotted for GRB2 (loading control) (right panel). The use of multi- strip Western procedure eliminated the need to strip the membrane or load another set of sample just to detect the signal of some house-keeping protein or non-phosphorylated ERK. Zone 6 protein GRB2 (right panel), which was obtained from the same gels and transferred on to another single membrane, served as corresponding loading control and was used to quantify the relative signal of phosphorylated ERK. Consequently, the time-course curves of activated ERK could be compared in a single graph (not shown).

[0158] Multi-strip Western blotting facilitates the detection of a large number of successive time-course data points without losing quality of the signal. With the help of this procedure, the time-course of protein activation in response to a specific stimulus may be markedly extended to include up to one hundred unique or repeated time-points (Fig. 3). Up to fifty data points in a time-course may be obtained if the experiment is designed to test an effect of a single perturbation, up to thirty three or twenty five - for two or three perturbations, respectively, in each experiment.

[0159] Fig. 6B represents an experiment, where the detailed time-courses of EGF-induced ERK activation had to be measured and compared in the absence or presence of PI3K inhibitor wortmannin (total - 24 data points). The comparison is more reliable, when a post-perturbation sample lies next to the control sample in a gel. To compare all 24 samples by conventional procedure, we would have to partition the sequence of data points, load them separately on to three mini-gels (8 samples per one gel) and transfer on to three different membranes. However, the data obtained from these membranes, could not be plotted in a seamless time-course graph. On the contrary, multi-strip Western blotting procedure allowed the reassembling of all data points on to one membrane for simultaneous exposure.

[0160] In Fig. 6B, HEK293 cells were starved overnight, treated with DMSO (W-) or 100 nM of wortmannin (W+) for 20 minutes, stimulated with 1 nM EGF for 0.5, 1, 2, 3, 4, 5, 7.5, 10, 15, 20, 30 minutes or left unstimulated (0 min), and lysed as described under section "Materials and Methods". The sample pairs of time points from 0 to 2 min were directly loaded on to Novex 4- 12% gradient Bis-Tris 10-well mini-gel A, from 3 to 7.5 min - on to gel B, and from 10 to 30 min - on to gel C. Proteins were resolved by LDS-PAGE under reducing conditions. Zone 4 and zone 5 strips were cut out of each gel, according to the migration of 10 μl Precision Plus Protein standards marker (M), assembled on to filter paper, covered with one nitrocellulose membrane and electroblotted. After transfer, the BSA-blocked membrane was cut into two pieces. The piece containing zone 4 proteins was immunoblotted for both phospho-AKT and phospho-SHP2 using the mixture of respective polyclonal antibodies (right panel). The piece with transferred zone 5 proteins was immunoblotted for phospho-ERKl/2 (left panel). Several proteins were detected after just one transfer cycle: phosphorylated ERK from zone 5 (Fig. 6B - left panel), 6OkDa phosphorylated AKT, a substrate of PI3K and a suitable readout of PI3K activity, from zone 4, and 70 kDa tyrosine phosphatase SHP2, used as loading control (Fig. 6B - right panel). [0161 ] There are situations, where it is impossible to separate some proteins in gels due to the close migration of these proteins in the same narrow zone. In this case, the inventors use the strategy of simultaneous detection: a single strip, containing both proteins, is cut out of each gel and transferred on to the same membrane, which is later incubated in a mixture of primary antibodies such as antibodies against phospho-AKT and antibodies against phospho-SHP2 (Fig. 6B - right panel).

[0162] The time interval between independent experiments may be very long. In most cases, data from one experiment needs to be analyzed prior to designing the next experiment, which may be performed under different conditions and then compared with the former. In this case, it is problematic to integrate independently measured data. We suggest a novel multi-strip-based approach for the integration of distinct data sets (Fig. 7)

[0163] Fig. 7 A is an example of multi-strip Western blotting application for the simultaneous (synchronous) comparison of data from several independent experiments. A549, T24, HEK293 or MCF-IOA cells were grown in 60 x 15 mm tissue culture plates until reached 80% confluence. Starved cells were stimulated with 0.1, 1 and 10 nM of EGF for 5 minutes or left untreated (control) and lysed. Gel A was loaded with straight A549 and reversed T24 sample sets. Gel B was similarly loaded with sample sets prepared from HEK293 and MCF-IOA cells. The multi-strip immunoblotting procedure was performed as described above to visualize p- AKT and α-tubulin (loading control). In this case, the cell lysates after each previous experiment are simply frozen and stored until all the lysates are prepared. At that time, all samples are prepared in Laemmli buffer and processed together. [0164] The principle of separate (asynchronous) comparison of the data from several independent experiments is shown on the Fig. 7B. Starved T24 or HEK293 were stimulated with indicated concentrations (nM) of IGF-I, EGF and HGF for 5 minutes and lysed in two separate experiments. In each case, the proteins were separated by LDS-PAGE. For standard curve (STD - upper right panel), a total of 20 ng active recombinant MAP kinase protein (aERK2) per first lane was loaded using 20 μl of 1 ng/μl protein Serial dilutions (1:2) were loaded on to subsequent lanes. The zone 5 gel strip (containing p-ERK) was placed on to assembling filter paper together with zone 4 strip (recombinant p-ERK migrates as 68kDa). Proteins were simultaneously transferred on to single membrane and probed for phospho-ERK (upper left panel). Charts show the differences of ERK phosphorylation in response to various stimuli between HEK293 (black solid columns) and T24 (diagonal striped columns) cell lines (Fig. 7D and E). Lower left chart was generated using raw signal intensities from two independent experiments (Fig. 7D), and lower right chart was constructed after conversion of raw signal intensities into absolute protein amounts using the coefficient (Fig. 7E), derived from standard calibration curves from separate experiments (Fig. 7C). Absolute protein amounts were divided by appropriate total protein amounts. In the preliminary experiments, a set of different dilutions of a chosen recombinant protein should be selected to provide linear range of signal intensities for the given conditions (including antibodies, substrate, exposure time, detection instrument etc.) and the detection method of choice. These dilutions should be used throughout all measurements. In our chemiluminescent signal detection system the linear range of the signal was estimated to lie between 0.150 and 20 ng of protein per lane.

[0165] A gel strip, containing the set of chosen dilutions, is combined on to an assembling filter and transferred on to the same membrane together with other gel strips that contain samples under investigation. Post detection, the signal intensities of recombinant protein bands are used to generate a standard calibration curve. Thus separate protein detection will result in calibration curve with individual slope. Upper right panel in Fig. 7B compares calibration curves from two independent experiments. Individual calibration curves are used to convert raw signal intensities of appropriate data (lower left panel) into the standardized protein amounts (ng of protein-of-interest per μg of total protein in lysate) (lower right panel). The use of the same dilutions of recombinant proteins eliminates the variation in immunoblotting conditions and instrumental detection from one experiment to another and provides an absolute scale for more accurate comparison of data from two or more different experiments.

[0166] With multi-strip Western blotting the experimental design becomes more flexible. A single membrane, containing strips from multiple gels, can reveal changes in protein activation and/or concentration upon multiple input factors: type and concentration of ligands, duration of the stimulus, presence of inhibitors or activators, blocking peptides, changes in expression levels of endogenous protein etc. The modification of Western blotting procedure described here meets the requirements of rapid, economical and productive data processing, which maintains high sensitivity and improves the accuracy of immunoblotting, which is critical in proteomics and protein biochemistry studies. EXAMPLE 2

Detailed protocol of the multi-strip Western blotting procedure [0167] Materials

[0168] For sample preparation, use sample buffer: 4x NuPAGE LDS Sample Preparation Buffer (pH 8.4) and 10x NuPAGE Sample Reducing Agent (both from Invitrogen, Carlsbad, CA). Alternately, Laemmli instead of LDS sample buffer can be used with appropriate running and transfer buffers. Electrophoresis can be performed under reducing as well as non-reducing conditions.

[0169] For SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE), one has to choose the type of apparatus for electrophoresis and protein transfer according to the size of your gels (e.g. mini- , midi- or maxi-gels). This protocol can be adapted for gels of any percentage, composition, size and with any number of wells. Examples include but are not limited to XCeIl SureLock Mini- Cell units (Invitrogen) using the NuPAGE Novex 4-12% gradient Bis-Tris Mini-gels (Invitrogen).

[0170] Running buffer: 50 mM 3-(N-morpholino)-propanesulfonic acid (MOPS), 50 mM Tris Base, 0.1% (w/v) sodium-dodecyl sulphate (SDS), 1 mM ethylene-diamine-tetraacetic acid (EDTA, pH 7.7) (available from Invitrogen and Boston Bioproducts, Worcester, MA). Store at room temperature. Supplement the running buffer in the upper chamber of XCeIl SureLock Mini-Cell with 0.5 ml NuPAGE Antioxidant (Invitrogen) before electrophoresis. Similarly, Laemmli gel running buffer can also be used when Laemmli sample buffer is used. [0171 ] Prestained molecular weight markers: Precision Plus Protein standards (Bio-Rad,

Hercules, CA). Store at -20⁰C.

[0172] For Western blotting, one has to choose the type of apparatus for electrophoresis and protein transfer according to the size of your gels (e.g. mini-, midi- or maxi-gels). For example, the XCeIl II Blot module (Invitrogen) used. The setup buffer: 20 mM Tris Base, 154 mM glycine, 0.02 % SDS, 20% (v/v) methanol, and the transfer buffer: 25 mM Bicine, 25 mM Bis-

Tris, 1.0 mM EDTA, 0.05 mM Chlorobutanol, pH 7.2 (available from Invitrogen), 10% (v/v) methanol. Store at 4°C. Supplement with 0.1% (v/v) NuPAGE Antioxidant (Invitrogen) in the transfer apparatus before electrophoretic transfer. Nitrocellulose membrane is from Bio-Rad.

Alternately, PVDF or nylon membranes can be also used. Blotting filter paper is at least 2.45 mm thickness, and 320 grade, and is available from E&K Scientific (Santa Clara, CA) or

Colonial Scientific (Richmond, VA).

[0173] For immunostaining, the Tris-buffered saline with Triton X-100 (TBS-T) buffer (IX) is

10 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.5% (v/v) Triton X-100. Store at room temperature.

Blocking buffer: dissolve 4% (w/v) heat inactivated bovine serum albumen (BSA) (Roche

Diagnostics, Indianapolis, EN) in TBS-T buffer. Primary and secondary antibodies diluted in

TBS-T buffer. Chemiluminescent reagent: SuperSignal West Dura Extended Duration Substrate

(Pierce Biotechnology, Rockford, IL). Square Dishes with Grid (Fisher) are useful for incubating the blot with the respective antibodies and for washing the blots.

[0174] Data acquisition can be performed using an Imaging system, such as the Image Station

440CF (Eastman Kodak Scientific Imaging Systems, New Haven, CT)

[0175] Methods

[0176] Preparation of Samples for Multistrip Western Blotting

[0177] Immediately after cell lysis, the supernatant of each cell lysate is mixed with 4x

NuPAGE LDS Sample Buffer and supplemented with 10x NuPAGE Sample Reducing Agent in a ratio of 65:25: 10 in labeled Eppendorf tubes. The tubes with samples are then heated for 5 min at 75°C. After cooling to room temperature, they are ready for immediate separation by SDS-

PAGE or can be stored for further use at -80⁰C.

[0178] Sample loading and SDS-PAGE

[0179] These instructions assume the use of XCeIl SureLock Mini-Cell apparatus for SDS-

PAGE of 10-well mini-gels. This protocol can be adapted for gels of any percentage, composition, size and with any number of wells. The number of gels to be loaded depends on the number of data series and the number of samples within each series to be analyzed. For example, when the time-course (e.g. 0, 1, 3, 5, 7, 10, 20, 30 and 60 minutes) of protein X activation in control cells (A) is compared to that in the presence of first perturbation (e.g. inhibitor of protein X) (B) and the second perturbation (e.g. the suppression of protein Y by siRNA) (C), one will have to load three data series (A, B and C) consisting of 9 time-points each

(Al, A2...A9 etc.) into three gels. There are two alternative ways of loading such array of samples (Fig. 13).

[0180] The upper chamber is filled with 200 ml of running buffer to completely cover the sample wells of a gel. 600 ml of running buffer is poured into the lower chamber.

[0181] A pipette equipped with prolonged gel loading tip (Fisher) is used to underlay 7 μl of prestained protein molecular weight marker (M) into the first and/or the last gel well.

[0182] Equal volume of each sample (e.g. 20 μl) is loaded into the rest of gel wells. The samples to be loaded can be different or repetitive. If there are empty wells without loaded sample left, fill them with similar amount of sample buffer. Mark the sequence of loaded samples in a laboratory notebook.

[0183] Lock the gel tension lever. The electrophoresis unit is then completely assembled by adding the lid on the buffer core, and connected to a power supply. The proteins are electrophoretically separated at 140 V, until the blue dye front reaches the bottom of a gel. If running more than 2 gels, make an interval of 5 minutes before loading the next tandem of gels and powering on the electrophoresis unit. This will reserve enough time for follow-up steps.

[0184] At the end of gel run, a gel cassette is removed out of apparatus and gently opened with a gel knife. Note that upon opening the cassette, the gel can be adhered on either side. If the gel remains on a notched side, the sequence of sampling should be rewritten in the laboratory notebook in a reversed order. Discard the plate of gel cassette without the gel.

[0185] The above gel loading and gel running steps can be performed with gel cassettes from other electrophoresis units.

[0186] Gel cutting

[0187] Fig. 2B illustrates the plate with attached gel after protein separation according to their molecular weight by SDS-PAGE. The prestained marker is visibly separated into the bands corresponding to protein molecular weights of 250, 150, 100, 75, 50, 37, 25, 20, 15 and 10 kDa

(Fig. 14, M).

[0188] A millimeter-scaled transparent ruler is firmly positioned near the edge lane with separated marker so that zero (0 cm) aligns with the middle of the blue dye front (Fig. 2B, BDF).

The distance from the blue dye front to the center of each marker band (Fig. 2B, H) is measured in millimeters and registered in a table. The table is designed to track the statistics of marker migration in the gel of selected percentage. In addition, different tables can be created according to the marker type used. The statistics is required for successive Multistrip Western blotting procedures if one needs to cut out the gel strip with the protein of interest (with known molecular weight), but no prestained marker has been loaded.

[0189] The distance between two electrophoretically separated marker bands corresponds to the migration range of certain molecular weight proteins. For instance, the distance between H250 and H150 defines a migration range of electrophoretically separated proteins with molecular sizes between 150 and 250 kDa. This range is termed zone 1 in the Fig. 2B. Each sample provides up to nine protein-containing zones that may be simultaneously cut out from a single gel.

[0190] A regular gel knife is used to cut out the strip, which covers an area with a protein of interest located in the middle, from the gel across its entire width (Fig. 2B, scissors symbol). [0191 ] The number of strips to be cut out from the gel depends on the number of distinct proteins to be detected. Most frequently studied signal transduction proteins migrating in various zones are listed in Table 1 , which also indicates the appropriate areas that can be cut out of the gel in order to detect these proteins later. For example, it is convenient to separate the activated EGF receptor (165 kDa, zone 1), from the phosphorylated PLDl (116 kDa) and the phosphorylated 90 kDa ribosomal S6 kinase (RSK) that migrate in the zones 2 and 3, respectively. Then it is easy to separate the phosphorylated Akt (60 kDa), which is found in the zone 4, from both the activated ERKl (44 kDa) and ERK2 (42 kDa) kinases that co-migrate in the zone 5, and from the phosphorylated S6 Ribosomal Protein (32 kDa, zone 6). In this case, according to the Table 1, the gel is cut into six strips at 12 mm and 22 mm (for phospho-S6 Ribosomal Protein), at 29 mm (for ERK1/2), at 38 mm (for phospho-Akt), at 44 mm (for phospho-RSK), at 49 mm (for phospho-PLDl) and at 54 mm (for phospho-EGFR) from the BDF.

[0192] If a protein of interest migrates in an intersection of marker-defined zones (e.g. 100 kDa GABl, which migrates between zones 2 and 3; 25 kDa Grb2, which migrates between zones 6 and 7; 74 kDa c-Raf, which migrates very close to the zone 3 etc.), the cutting area must include both zones or at least should be wider. For example, She protein has three isoforms of 46 kDa, 52 kDa and 66 kDa migrating in the zones 4 and 5, so it can be separated from the proteins that lay in the zone 3 (e.g. p85, a regulatory subunit of PI3K) and between zones 6 and 7 (e.g. GRB2) by cutting the gel into three strips at 9, 22, 38 and 49 mm (see Table 1). [0193] The gel pieces outside the strips are discarded.

[0194] The first plate with prepared multiple gel strips is covered with a sheet of moistened filter paper (CFP, for covering filter paper) and placed on the bench top. Similarly, the second gel is cut, covered with another sheet of moistened CFP and placed next to the previously laid plate on the bench top. Repeat above procedure with the rest of the gels. [0195] Assembly of gel strips

[0196] During this step, the gel strips that are derived from different gels, are assembled onto a single sheet of filter paper (AFP, for assembling filter paper) for the subsequent electrophoretic protein transfer onto the same piece of nitrocellulose membrane. The maximal number of gel strips that can be combined onto a single AFP depends on the overall dimension of the transfer unit, hence on the size (height and width) of AFP. Routinely we use 7x10 cm filter, which provides space for maximum of twelve gel strips of 0.6 cm height each. However, regularly we place fewer amounts of gel strips (e.g. six), especially when they are wider and/or the membrane should be cut into two or more pieces after electrophoretic protein transfer. The strategy of assembly depends on the quantity of gels used as well on the number of analyzable proteins per lane (i.e. the number of precut gel strips comprising of appropriate zones). Here we provide two exemplar cases of gel strip assembly.

[0197] Case 1. When six gels (A, B, C, D, E and F) are run and five proteins of interest from each sample (e.g. phospho-EGFR from zone 1, phospho-GABl migrating between zones 2 and 3, phospho-SHP2 from zone 4, phospho-ERKl/2 from zone 5 and Grb2 as house-keeping protein, which migrates between zones 6 and 7) are subsequently detected under equal conditions. Guidance for cutting of one out of six gels is provided in left panel of Fig. 14. [0198] Case 2. When three gels (A, B and C) are run and four proteins from each sample (phospho-IRS 1 from zone 1, phospho-IR from zone 3, phospho-Akt from zone 4 and GAPDH as house-keeping protein, which migrates between zones 5 and 6) are analyzed. Guidance for cutting of one out of three gels is provided in right panel of Fig. 14.

[0199] In case 1, the plate containing gel A strips is flipped and gently lifted so that all strips would stick to the moistened CFP. Use gel knife if the strips do not independently detach from the plate.

[0200] The first gel strip from the top (possessing zone 1 proteins) is lifted with a gloved hand and carefully transferred onto the AFP #1. The CFP with remaining gel strips is returned onto the plate by flipping it back.

[0201 ] Similarly, top gel strips derived from gels B to F are sequentially transferred onto the AFP #1 so that the strips would lay side by side and parallel to each other. AFP #1 is now ready for immediate protein transfer. If some pauses occur, regularly wet the surface of gel strips by dropping deionized water. If some pauses occur, regularly wet the surface of gel strips by dropping deionized water. [0202] The previous three steps are repeated with the strips derived from gels A to F that possess the proteins migrating in zones 1, 2+3, 4, 5 and finally 6+7. This procedure will yield five AFPs (AFP #1 through #5) with collected six gel strips on each (Fig. 15A). Now they are ready for electrophoretic protein transfer onto the same membrane.

[0203] In case 2, similar gel lifting onto moistened CFP and strip lifting onto the AFP are performed with gel A, followed by sequential transfer of gel strips derived from gel B and gel C onto the AFP #1. Then, gel strips that contain the proteins migrating in zone 3 are sequentially placed onto the AFP #1 below previously laid triplet of strips. Leave a small gap between triplets.

[0204] AFP #2 is processed in the same manner so that it would contain triplet of strips with zone 4 and triplet of strips with zone 5+6 (Fig. 15). After protein transfer, the resulting nitrocellulose membrane is cut into two pieces across the gap between triplets. The pieces are then treated in separate dishes. Alternatively, the whole piece of nitrocellulose membrane can be treated with blocking reagent and then incubated with the mixture of primary antibodies (be sure that they do not cross-react) in a single dish.

[0205] Western blotting

[0206] Instructions provided below assume the use of XCeIl II Blot module that is used for protein transfer from one AFP.

[0207] Onside of assembly tray is filled with 500 ml of setup buffer, while another side - with

400 ml of transfer buffer.

[0208] Four sponge pads are presoaked in setup buffer. Remove air bubbles by squeezing the pads while they are submerged in buffer. A nitrocellulose membrane is cut to the dimensions of

AFP and presoaked in transfer buffer for 5 minutes before using. Three additional sheets of filter paper are briefly moistened in setup buffer immediately before using.

[0209] Two soaked sponge pads are placed into the cathode (-) core of the blot module and covered with one sheet of moistened filter paper. The AFP with collected gel strips is placed on the top. Subsequently, the surface of gel strips is covered with a sheet of nitrocellulose membrane. Remove any trapped air bubbles by rolling a blotting roller over the membrane surface. Two moistened filters are then placed onto the surface of the membrane followed by tandem of soaked sponge pads. The pads should rise at least 0.5 cm over the rim of the cathode core. If not, place an additional filter paper of sponge pad in the tank.

[0210] The anode (+) core is placed on the top of the pads. Slide the blot module into the rails on the lower chamber. Lock the gel tension lever. [021 1 ] The blot module is filled with transfer buffer until the blotting sandwich is completely submerged. The outer chamber is filled with cold deionized water.

[0212] The unit is completely assembled by adding the lid on the buffer core, and connected to a power supply. The proteins are electrophoretically transferred at 30 V constant for 90 minutes.

[0213] After transfer is stopped, the nitrocellulose membrane is removed out of the blot module and placed into a square Petri dish. Used filter papers and gel strips are discarded.

[0214] After 3 minute rinsing with deionized water, the membrane is incubated with 20 ml of blocking buffer for 1 hour at room temperature on a rotating platform.

[0215] After the blocking buffer is discarded, the membrane is briefly rinsed with deionized water and blotted with appropriate primary antibody at dilution ratio as recommended by a manufacturer overnight at 40⁰C on a rotating platform. The primary antibodies can be collected into the tube and reused several times if supplemented with 0.1% sodium azide. If precipitation occurs, filter the solution through 0.22 μm filter unit.

[0216] The membrane is extensively rinsed with deionized water and washed five times for 7 minutes each with TBS-T buffer at room temperature on a rotating platform.

[0217] The membrane is incubated with appropriate secondary antibody at dilution ratio as recommended by a manufacturer for 1 hour at room temperature on a rotating platform followed by step 10 once again.

[0218] The membrane is incubated with a working solution of chemiluminescent reagent for 5 minutes and the signal is captured by Imaging system and quantified using KODAK Digital

Science software. The chemiluminescent signal can be visualized by another imaging instrument and quantified using an appropriate software. Alternatively, the signal can be captured on the film followed by densitometric quantification. To enable side-by-side comparison, the capture time and number of frames should be equal for each separately exposed membrane.

EXAMPLE 3

[0219] PreCut'n'cast gels

[0220] The inventors has develop a special precast gel, which would already contain pre- separated gel strips according to molecular weights of proteins of interest. These gels are referred to as PreCut'n'Cast gels. The positions of the separation lines depends on the molecular weight (in kDa) of the protein of interest. The molecular weight of a great number of proteins have been determined empirically. These include, but are not limited to, signaling proteins of the Wnt/D-catenin signaling pathway, the TGFD- signaling pathway, the inflammation (NFkB) signaling pathway, the TCR/BCR signaling pathway, the death receptor/apoptosis signaling pathway, the mitogenic and integrin signaling pathway, and stem cell markers. The molecular weights of these proteins are shown in Tables 2-9. Using these tables, one can select a PreCut'n'Cast gel with the ideal gel percentage and the zonal strips. [0221 ] There are at least three different procedures to generate PreCut'n'Cast gels that contain multiple slices. These gels include polyacrylamide gels and agarose gels. [0222] In the first procedure of developing PreCut'n'Cast gel, a solidified gel of a given polyacrylamide density is cut into the strips using a sharp blade in horizontal or vertical setting. The gel cassette is closed afterwards. Importantly, the cutting procedure does not affect the migration of proteins in the strip gels, as presented in Fig.16.

[0223] The second and third procedures of making PreCut'n'Cast gels includes the preparation of both fixed (e.g. containing 8%, 10% or 12% polyacrylamide etc.) and gradient (e.g. from 4 to 12% polyacrylamide) PreCut'n'Cast gels.

[0224] In the second procedure, the gel strip are prepared in a vertical or horizontal cassette. Each strip is separated from the neighboring slice by a narrow region of a low density polyacrylamide. Specifically, starting from the bottom, the sufficient volume of liquid polyacrylamide (e.g., 5 ml) of fixed density and greater than 4% (e.g., 8%, 10% or more) is poured into the cassette for the first (bottom) strip. Polyacrylamide solution contains gel strengthener additive (e.g. Rhinohide, Invitrogen), which makes gel much stronger. After it solidifies a small amount of lower density polyacrylamide (e.g., 3 or 4%; without strengthener), which is able to solidify, is poured to form a narrow (1-2 mm) boundary, separating two neighboring strip. The procedure is repeated to form the next strip until as many as nine strip for different molecular weight proteins are formed (Fig. 12).

[0225] In the third procedure, the gel strip are prepared in a similar manner to second procedure, but they are separated by a thin strip of plastic film. Such film is solid but porous and transparent for protein migration in the electrical field. Thereby the film allows separating the neighboring gel slices after polyacrylamide gel electrophoresis (PAGE) under native or denaturing conditions. Moreover, these films can be exploited to separate different slices following the opening of the gel cassette if one side of the film surface is treated in order to attach to the polyacrylamide gel, and another side is not. Such films are readily available (e.g. polyester NetFix film from Serva).

[0226] In another embodiment, this invention provides devices to cut and assemble gel strips according to the method described herein. To meet versatile needs of consumers, the devices can be used on both large company and small academic laboratory scales and thus have different levels of sophistication. [0227] The PreCut'n'Cast gels including the devices for their preparation can be readily manufactured to save money and time for quantitative Western blot measurements.

[0228] The references cited herein and throughout the specification are incorporated herein by reference.

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Table 1. Migration of various signal transduction proteins in Novex 4-12% gradient NuPAGE mini-gel and their cutting areas, based on the migration of prestained Precision Plus Protein marker (BIO-RAD).

£

Table 2. Possible casting and pre-determination of the zones for PreCut'n'Cast gels applicable for studying Wnt/β-Catenin Signaling. Table 2 also shows the molecular sizes of proteins found in the Wnt/D-Catenin Signaling pathway.

Table 3. Possible casting and pre-determination of the zones for PreCut'n'Cast gels applicable for studying TG Fβ signaling pathway. Table 3 also shows the molecular sizes of proteins found in the TGFβ signaling pathway.

Table 4. Possible casting and pre-determination of the zones for PreCut'n'Cast gels applicable for studying the inflammation (NFKB) pathway. Table 4 also shows the molecular sizes of proteins found in the inflammation (NFKB) pathway.

* - Ab from Cell Signaling

Table 5. Possible casting and pre-determination of the zones for PreCut'n'Cast gels applicable for studying the TCR/BCR signaling pathway. Table 5 also shows the molecular sizes of proteins found in the TCR/BCR signaling pathway.

Table 6. Possible casting and pre-determination of the zones for PreCut'n'Cast gels applicable for studying the death receptor/apoptosis signaling pathway. Table 6 also shows the molecular sizes of proteins found in the death receptor/apoptosis signaling pathway.

Table 7. Possible casting and pre-determination of the zones for PreCut'n'Cast gels applicable for studying the mitogenic and integrin signaling pathway. Table 7 also shows the molecular sizes of proteins found in the mitogenic and integrin signaling pathway.

Migration range of signal transduction proteins in the gel

Protein

PreCut'n'Cast PreCut'n'Cast PreCut'n'Cast PreCut'n'Cast

Zone MW

Gel Gel Gel Gel (kDa) MAPK #1 MAPK #2 MAPK #3 MAPK #4

SOS1/2 (185,

155 kDa)

EGFR (175

SOS 1/2 (185, kDa) SOS1/2 U85,

SOS1/2 (185, 155 kDa) PDGFα/β (195, 155 kDa)

155 kDa) EGFR (175 19O kDa) EGFR (175

EGFR (175 kDa) ErbB2/3/4 kDa) kDa)

PDGFα/β (195, (185, 18O kDa) PDGFα/β (195,

PDGFα/β (195, 190 kDa) VEGFR2 (210, 190 kDa)

19O kDa) ErbB2/3/4 230 kDa) ErbB2/3/4

ErbB2/3/4 (185, (185, 18O kDa) VEGFR3 (195 (185, 18O kDa)

18O kDa) VEGFR2 (210, kDa) VEGFR2 (210,

VEGFR2 (210,

160- 230 kDa) RhoGAP (190 230 kDa)

230 kDa) 260 VEGFR3 (195 kDa) VEGFR3 (195

VEGFR3 (195 kDa) c-Ret (170, 175 kDa) kDa)

RhoGAP (190 kDa) RhoGAP (190

RhoGAP (190 kDa) M-CSFR (175 kDa) kDa) c-Ret (170, 175 kDa) c-Ret (170, 175 c-Ret (170, 175 kDa) TSC2 (200 kDa) kDa)

M-CSFR (175 kDa) M-CSFR (175

M-CSFR (175 kDa) SHIP2 (160 kDa) kDa)

TSC2 (200 kDa) TSC2 (200

TSC2 (200 kDa) kDa) SHIPl (145 kDa) kDa) c-Cbl (120 kDa)

GABl (105-110 kDa)

PIlO-PDK(IlO kDa)

PTP-PEST

(110-125 kDa) cPLA2 (110

SHIPl (145 kDa)

SHIPl (145 kDa)

SHIPl (145 kDa) c-Cbl (120 kDa) c-Cbl (120 kDa) c-Cbl (120 kDa) kDa) FGFR (120,

FGFR (120, 145

FGFR (120, 145 kDa) kDa)

145 kDa) c-Met(145 c-Met(145 c-Met(145 kDa) kDa) kDa) PLC (150-155

PLC (150-155

110- PLC (150-155 kDa) kDa) 160 kDa) p 120 RasGAP p 120 RasGAP p 120 RasGAP (120 kDa)

(12OkDa)

(120 kDa) FAK (125

FAK (125 kDa)

FAK (125 kDa) kDa)

Cas (130 kDa)

Cas (130 kDa) Cas (130 kDa)

PLD1/2(117,

PLD1/2(117, PLD1/2(117,

12OkDa)

120 kDa) 12OkDa)

Pyk2(116kDa)

Pyk2(116kDa) Pyk2(116

KSR (115 kDa)

KSR (115 kDa) kDa)

InsRβ (95 kDa)

KSR (115 kDa)

IGF- lRβ (95 kDa) p85-PI3K (85 kDa) p90 RSK (90 kDa)

GAB2 (98 kDa)

Table 8. Possible casting and pre-determination of the zones for PreCut'n'Cast gels applicable for studying the stem cells. Table 8 also shows the molecular sizes of stem cell markers.

Table 9. Possible casting and pre-determination of the zones for PreCut'n'Cast gels applicable for studying the mitogenic and integrin signaling pathway. Table 7 also shows the molecular sizes of proteins found in the mitogenic and integrin signaling pathway

Claims

What is claimed:

1. A method for increasing quantitative data output of biological molecules from multiple samples comprising: a) performing gel electrophoreses on a plurality of same sized gels; b) electrophoretically resolving each gel under conditions to result in substantially identical separation of molecular markers on the gels; c) dividing each gels into strips that correspond to a set of pre-determined zones based on the location of the molecular marker on the gels; and d) arranging the strips from equivalent zone from each gel in parallel tandem with each other and in contact a filter paper to form a multi-strip gel.

2. A method of claim 1, wherein the biological molecule is protein, RNA, or DNA.

3. A method of claim 1, wherein the same sized gels are polyacrylamide gels.

4. A method of claim 1, wherein the same sized gels are agarose gels.

5. A method of claim 1, wherein the same sized gels are precast gels.

6. A method of claim 5, wherein the precast gels comprise gel strips.

7. A method of claim 1, wherein the same sized gels are gradient polyacrylamide gels.

8. A method of claim 1, wherein the same sized gels are single percentage polyacrylamide gels.

9. A method of claim 1, wherein the same sized gels are the second dimensional vertical slab polyacrylamide gels of two-dimensional gel electrophoresis.

10. A method of claim 1, wherein the molecular markers are pre-stained.

11. A method of claim 1, further comprising electro-transferring the multi- strip gel on to a membrane.

12. A method of claim 1, wherein the same sized gels are selected from a group consisting of: a) mini gels of 8 X 8 cm; b) mini gels of 8.6 X 6.8 cm; c) midi gels of 13 X 8.3 cm; d) mini gels of 9 X 6 cm; e) mini gels of 10.5 X 6 cm; f) mini gels of 12.5 X 6 cm; g) large gels of 20 x 20 cm.

13. A method of claim 1, wherein the plurality of same sized gels is from 2 to 12.

14. A method of claim 11, where in the electro-transfer membrane is nitrocellulose, PVDF, or nylon.

15. A method of claim 11, further comprising processing the membrane for Western blot analysis and protein quantification.

16. A method of claim 1, wherein the multi-strip gel is dried.

17. A method of claim 16, wherein the dried multi-strip gel is exposed to autoradiograph films or storage phosphor screens.

18. A method of claim 1, wherein the multi- strip gel is stained with nucleic acid stains.