WO2007010532A2 - Compositions for one step colorimetric analysis and methods of using same - Google Patents

Compositions for one step colorimetric analysis and methods of using same Download PDF

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Publication number
WO2007010532A2
WO2007010532A2 PCT/IL2006/000830 IL2006000830W WO2007010532A2 WO 2007010532 A2 WO2007010532 A2 WO 2007010532A2 IL 2006000830 W IL2006000830 W IL 2006000830W WO 2007010532 A2 WO2007010532 A2 WO 2007010532A2
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Prior art keywords
staining
composition
porous
gel
solution
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PCT/IL2006/000830
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French (fr)
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WO2007010532A3 (en
Inventor
Shmuel Bukshpan
Gleb Zilberstein
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Shmuel Bukshpan
Gleb Zilberstein
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Publication of WO2007010532A2 publication Critical patent/WO2007010532A2/en
Publication of WO2007010532A3 publication Critical patent/WO2007010532A3/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins

Definitions

  • the present invention relates to compositions, kits and methods for identifying and visualizing biological moieties in a sample.
  • Coloi ⁇ metric methods are commonly used after electrophoretic separation of proteins or other bioparticles, such as polynucleotides. These methods are an essential step in the process of identifying the content of biological samples.
  • the principle of such colorimeti ⁇ c methods is to provide a visible indication of the species separated along the gel, by using staining agents that selectively form complexes with said species and less selectively form complexes with the surrounding gel.
  • colorimetric methods are also used as an initial purification step.
  • staining protocols Numerous staining protocols, staining agents and ready-to-use compositions are commercially available and are generally utilized for colorimetric methods.
  • the most common staining procedures include CoomassieTM Blue, Silver or fluorescent Sypro® Ruby, Ponceau, fluorescamine®, Light Green, Fast Green, Amido Black or a mixture thereof (Romano and Wirth, J. Chromatography, 698: 123-143, 1995).
  • US Patent No. 4,219,337 discloses an assay for proteins or polypeptides in solution using Coomassie Brilliant Blue G250 dye in perchloric or hydrochloric acid. Upon mixture of the acidic reagent with a protein sample, the dye couples with the protein and undergoes a color change, following which the absorbance of the mixture can be measured to provide a quantitative analysis of the protein in the sample.
  • US Patent No. 4,839,294 discloses an assay useful for the evaluation of the lipid content of a biological fluid in aqueous dispersion.
  • the assay utilizes a dye-detergent complex which when solubilized by an aqueous dispersion of lipid-containing fluid sample, exhibits a characteristic color.
  • US Patent No. 6,057,160 discloses a method for making Coomassie Brilliant
  • CBB dye/protein complex comprising mixing a protein with a non-alcoholic aqueous CBB solution having a pH greater than 4, heating the mixture to a first temperature for a short pulse period, then cooling to a second temperature to form a solution containing the CBB dye/protein complex.
  • 6,277,643 discloses a method for the detection of protein on polyacrylamide gels comprising the steps of staining the polyacrylamide gels with a counter-dye composition containing an acidic organic dye and a basic organic dye, selected from the group consisting of Zincon and Ethyl violet, Zincon and Methyl violet, Zincon and Meldola's blue, Coomassie brilliant blue R and Phenosafranin, Coomassie brilliant blue G and Methyl orange, Calconcarboxylic acid and Rhodamine B, and Eriochrome black T and Rhodamine B and protein complexes with the counter- dye composition, and observing protein-counter-dye complexes.
  • a counter-dye composition containing an acidic organic dye and a basic organic dye, selected from the group consisting of Zincon and Ethyl violet, Zincon and Methyl violet, Zincon and Meldola's blue, Coomassie brilliant blue R and Phenosafranin, Coo
  • compositions, methods and kits comprising porous substrates, including beads, small particles, porous sheets and plates among others and staining compositions comprising one or more staining agents that form visible
  • the invention further provides methods for identifying biological moieties in a sample, using the compositions and kits of the invention.
  • the methods of the invention confer distinct advantages over colorimetric methods known in the art as they provide an exceptionally high signal to noise ratio of stained moieties in a gel. Due to the low background signal (noise), the methods of the invention generally do not require any destaining step as the signals obtained from the protein or other molecular species of interest are clear and can be analyzed or further purified, as is.
  • the methods of the invention utilize for the first time delivery means that discriminate with exceptional sensitivity between the protein bands incoiporated in the gel and the gel itself. According to this principle, the gel retains almost no staining agent and advantageously no destaining is required. In view of these advantages, the methods of the invention are significantly faster than conventional staining procedures, requiring only one step, which may take no more than several minutes. The one-step method is particularly simple and thus no particular training is required for its application.
  • the present invention provides a composition for staining a biological moiety comprising a porous substrate comprising at least one staining component capable of forming a visual signal upon contact with a biological moiety.
  • the staining component is absorbed onto the porous substrate.
  • the staining component is contained within the porous substrate. The porous substrate is insoluble in the surrounding medium into which the staining agents are released.
  • the retention or adsorption of the staining agent to the porous substrate is sufficient largely to prevent non specific background staining and yet to permit complex formation with the species of interest thereby increasing the signal to noise ratio.
  • the staining component comprises at least one staining agent.
  • the at least one staining agent is selected from the group consisting of: CoomassieTM Blue, Silver stain, Sypro® Ruby, Ponceau, fluorescamine®, Light Green, Fast Green, Amido Black, Ethydium Bromide and Propidium Bromide.
  • the staining component comprises a plurality of staining agents.
  • the porous substrate comprises a polymer, wherein the polymer is linear or cross-linked.
  • the porous substrate is selected from the group consisting of: porous bead, porous small particles, porous sheets, porous plates and porous fibers.
  • the composition is in a form of ready to use suspensions wherein the porous substrate particles are suspended in an appropriate solution. According to yet another embodiment, the composition is in a dry form.
  • the dry form composition is capable of being transformed into a ready-to-use form upon reconstitution in a carrier.
  • the carrier is selected from the group consisting of: a hydrophilic solution, a water based-solution, water and a buffer.
  • compositions of the invention may advantageously be reusable, inasmuch as only a relatively small amount of the total dye contained within the porous substrate is released during each staining procedure.
  • the present invention provides a kit comprising a first component comprising a staining component capable of forming a visual signal upon contact with a biological moiety and a second component comprising porous substrate.
  • the staining component is absorbed onto the porous substrate.
  • the staining component is contained within the porous substrate.
  • the staining component comprises at least one staining agent.
  • the at least one staining agent is selected from the group consisting of: CoomassieTM Blue, Silver stain, Sypro® Ruby, Ponceau, fluorescamine®, Light Green, Fast Green, Amido Black, Ethydium Bromide and Propidium Bromide.
  • the staining component comprises a plurality of staining agents.
  • the porous substrate comprises a polymer, wherein the polymer is linear or cross-linked.
  • the porous substrate is selected from the group consisting of: porous bead, porous small particles, porous sheets, porous plates and porous fibers.
  • the staining agent is in the form of a ready- to-use composition.
  • the staining agent is ready to use upon reconstitution in a carrier.
  • the kit further comprises a carrier.
  • the carrier is selected from the group consisting of: a hydrophilic solution, a water based-solution, water and a buffer.
  • the present invention provides a one-step method for staining at least one biological moiety in a gel, the method comprising contacting the gel with a staining composition comprising a staining component comprising at least one staining agent capable of forming a visual signal upon contact with a biological moiety and a porous substrate.
  • the method further comprises the step of incubating the composition with the gel until a visual indication is obtained.
  • the method further comprises the step of rinsing the gel with a rinsing solution.
  • the rinsing solution is selected from a group consisting of: a hydrophilic solution, a water based-solution, water and a buffer.
  • the contacting step includes immersing the gel in the composition by mechanical means.
  • the contacting step includes spraying the composition over the gel.
  • the present invention provides a method for staining at least one biological moiety in a gel, the method comprising: providing a composition comprising a staining component comprising at least one staining agent capable of forming a visual signal upon contact with a biological moiety and at least one porous substrate, wherein the staining component is contained within the at least one porous substrate; providing a gel containing biological moieties; and contacting the gel with the composition .
  • the staining composition comprises a plurality of porous substrates. According to some embodiments the staining composition comprises a plurality of staining agents.
  • the method comprises incubating the composition with the gel until a desired visual indication is obtained.
  • the method comprises rinsing the gel with a rinsing solution.
  • the rinsing solution is selected from a group consisting of: a hydrophilic solution, a water based-solution, water and a buffer.
  • contacting the gel with the composition is carried out by immersing the gel in the composition. According to yet another embodiment, contacting the gel with the composition is carried out by spraying the composition over the gel. According to yet another embodiment, contacting the gel with the composition is earned out by mechanical means.
  • the composition is prepared by a method comprising: providing a staining agent; providing a porous substrate comprising a plurality of porous beads; and mixing the staining agent and the porous beads; and incubating the mixture until at least a portion of the staining agent is adsorbed in the beads; and, optionally, rinsing the mixture with a rinsing solution.
  • the staining agent is prepared prior to being mixed with the porous substrate by solution or reconstitution in a suitable carrier.
  • the porous substrate is selected from but not limited to: porous bead, porous small particles, porous sheets, porous plates and porous fibers.
  • Figure 1 demonstrates SDS PAGE of protein bands stained by the method of invention on a 0.5 mm acrylamide gel.
  • Figure 2 presents lactogolobiilin protein bands on a 1.5 mm acrylamide gel.
  • Figure 3 shows the results obtained from staining protein mass markers (Biorad®) on a 0.5 mm gel.
  • Figure 4 shows staining obtained from 1 ng of protein of after 8 hr (A) and 15 hr (B) of incubation within the staining mixture.
  • Figure 5 demonstrates staining of protein spots after two weeks of incubation in the staining composition of the invention.
  • Figure 6 shows the results obtained from staining BSA bands, following the separation, using the Sypro® Ruby staining agent in a composition of the invention. The result was obtained after 30 minutes.
  • the principle of the staining composition of the present invention is that the staining solution is based on the incorporation of the dye within a porous matrix (e.g. beads, small particles, sheets and fibers).
  • a porous matrix e.g. beads, small particles, sheets and fibers.
  • the term 'beads' is used hereinafter to describe any suitable porous material, including sheets, fibers and small particles, which may be used in the context of the present invention. Since the dye is adsorbed in the beads and is held in the porous matrix by capillary forces, the diffusion process by which the dye can leak from the beads is quite slow. Therefore upon adding the composition of the invention to a solution or onto a gel only relatively very small quantities of the total dye escape the porous beads.
  • the present invention varies from the prior art staining compositions and methods in several respects.
  • the present invention is adapted to identify the content of a sample dispersed in a gel by a single step, namely the step of contacting the gel with the staining solution followed by an optional quick and simple rinsing of the gel after a few minutes of contact without the need for a destaining step.
  • using the compositions and methods of the present invention for protein identification does not require particular skills due to the simplicity of the process.
  • the present invention provides visible indication of the content of a biological sample with significantly high signal to noise ratio. In fact, the background signal is exceptionally low as compared to the background signals obtained with other colorimetric methods for non-specific identification of biological moieties, known in the art.
  • the present invention due to the outstanding signal to noise ratio which is obtained within several minutes, the present invention provides an extremely fast procedure for clear identification of various moieties in a gel.
  • porous substrate refers to any porous substrate, such as beads, fibers, sheets and small particles, capable of adsorbing a solution, particularly a solution comprising one or more staining agents.
  • the rate of diffusion of the staining agent out of the beads, towards an inert environment which is devoid of proteins and/or other biological moieties is sufficiently slow such that biological moieties (e.g., proteins, nucleic acids, etc.) are preferentially and selectively stained over the inert environment.
  • the porous substrate has the ability to release the dye within a gel containing biological moieties, such as a poly aery lamide gel or an agarose gel.
  • the porous substrate is typically formed from a polymer that serves as a support for some components of a reaction system.
  • it may contain an appropriate functional group that can be bound to chemical or biological moieties, e.g., by electrostatic or hydrophobic interactions, or may function as a reagent, catalyst and protecting function, etc.
  • the polymer can be linear or crosslinked.
  • the polymer will be suitable to form beads or other forms of porous substrates that are insoluble macroscopically in the medium to which they are exposed but can swell in an appropriate solvent.
  • porous beads that can be used in the context of the present invention are known in the art and certain beads are also commercially available. Such beads include beads obtained from phenolic complexes of water-insoluble but water-swellable crosslinked N-vinyl-lactam or N-alkyl-N-vinylamide polymers as disclosed in US
  • a porous, bead-like copolymer is disclosed in US Patent No. 4,686,243.
  • the bead is obtained by suspension polymerization of monomeric oxiranylalkyl compounds and/or 2-aziridinylalkyl compounds, n-valent crosslinking agents, N-vinylamides, heterocyclic 5-membered ring compounds or 6-membered ring compounds, having a polymerizable olefinic group in each case, and polymerizable quaternary ammonium salts.
  • a durable and sprayable controlled release bead of active ingredient in the pores of a polymeric micro-porous bead is disclosed US Patent No. 5,888,930.
  • the bead has an anisotropic pore structure of large pores in the interior and small pores at the surface, wherein the gradation of pore sizes between the interior and the surface being continuous.
  • US Patent No. 5,306,632 discloses a porous shaped substrate, such as a porous bead, having a hydrophilic surface containing amide groups constituting about 1.8 mole percent to less than about 15 mole percent of the total nitrile groups, and containing no amide or carboxyl groups.
  • the substrate is substantially non-swellable in water and is able to resist pressures in a columnar bed of up to about 3000 psi without collapsing.
  • an alkaline catalyst such as sodium hydroxide and a nonsolvent for the substrate such as methanol are combined to form a suspension.
  • a peroxide is added to the suspension and the suspension is heated to hydrolyze nitrile groups to amide groups.
  • Succinylated aminoethyl groups or activated carboxyl groups can be formed on the substrate and a bioactive ligand such as /> ⁇ aminobenzamidine can be covalently bound to the substrate.
  • the bound ligand can then be used to complex a biologically active substance such as an enzyme to separate the substance from solution.
  • a composition comprising a porous bead and a pigment for cosmetic applications is disclosed in US Patent No. 6,171,602. However, the composition is formulated to prevent bleeding of the pigment out of the bead and for this purpose the composition must contain a hydroalcoholic base.
  • the composition of the present invention is formulated to enable specific bleeding or release of the dye upon contact with biological moieties, but not upon contact with other materials particularly gels.
  • the porous bead for the composition of the invention may be purchased or prepared by any method known in the art.
  • US Patent No. 4,229,547 discloses a polymerization process which produces spherical beads of polymer having excellent porosity and unusually high bulk density. The process comprises polymerizing discrete droplets of liquid monomer containing a monomer-soluble free radical type catalyst while suspended in aqueous mucilage having plastic flow properties and in the presence of a nonionic surfactant having the proper Hydrophile-Lipophile Balance (HLB), such as, for example, sorbitan monooleate.
  • HLB Hydrophile-Lipophile Balance
  • Patent No. 5,369,133 The method comprises introducing a mixture comprising a purified monomer, initiator and pore-forming agent into a 4-necked reactor with a suspension stabilizer and aqueous solution, suspending the mixture to provide homogeneous organic droplets at a temperature sufficiently low so as not to decompose the initiator, increasing the temperature gradually to a final reaction temperature and maintaining the temperature at the final temperature to react completely by suspension- polymerization the monomers in the individual organic droplet to provide the porous polymer bead, separating the bead, washing the bead with water, acetone and tetrahydrofuran, successively, and drying the bead, precipitating the bead in a solution mixed with monomer and thinner to fill the pores of the porous bead with the diluted monomers, and introducing in a 4-necked reactor with a suspension stabilizer and aqueous solution, and by suspension-polymerization again, to coat the lightly cross- linked gel-type polymer on the surface of
  • biological moiety refers to either man-made or natural proteins, protein-DNA complexes, DNA, RNA, enzymes, peptides, polypeptides, antibodies, antigens, antigenic epitopes and variants thereof, polynucleotides, hormones, carbohydrates, lipids, phospholipids and biotinylated probes.
  • the bead based staining solution can be applied to the gel either by submerging the gel in a water suspension of the beads or by spraying the gel with the suspension of beads or by using a specially designed applicator.
  • This staining solution can be easily incorporated in any electrophoresis set up such as standard cassette designs among others.
  • the staining compound can be supplied in the form of a kit comprising two separate components, the dye as the first component and the beads at the second components.
  • the two components may be mixed extemporaneously at the time of use.
  • the present invention further provides a one-step method for staining at least one biological moiety in a gel, the method comprising contacting the gel with a staining composition comprising a staining component comprising at least one staining agent capable of forming a visual signal upon contact with a biological moiety and a porous substrate.
  • the method further comprises the step of incubating the composition with the gel until a visual indication is obtained.
  • the method further comprises the step of rinsing the gel with a rinsing solution.
  • the rinsing solution is selected from a group consisting of: a hydrophilic solution, a water based-solution, water and a buffer.
  • the contacting step includes immersing the gel in the composition by mechanical means.
  • the contacting step includes spraying the composition over the gel.
  • the invention further provides a method for staining biological moiety in a gel, the method comprising: providing a composition comprising a staining component comprising at least one staining agent capable of forming a visual signal upon contact with a biological moiety and porous substrates; providing a gel containing biological moieties; and contacting the composition with the gel.
  • the method comprising incubating the composition with the gel until a desired visual indication is obtained.
  • the method comprises rinsing the gel with a rinsing solution.
  • the rinsing solution is selected from a group consisting of: hydrophilic solution, water based-solution, water and a buffer.
  • contacting the composition with the gel is carried out by immersing the gel in the composition. According to yet another embodiment, contacting the composition with the gel is carried out by spraying the composition over the gel. According to yet another embodiment, contacting the composition with the gel is carried out by mechanical means, such as clamps.
  • the composition is prepared by a method comprising: providing a staining agent; providing porous substrates; and mixing the staining agent and the porous substrate; and incubating the mixture until at least a portion of the staining agent is adsorbed in the porous substrate; and, optionally, rinsing the mixture with a rinsing solution.
  • the staining agent is prepared prior to being mixed with the porous substrates by reconstitution in a carrier.
  • the kit of the invention may include a ready-to-use gel cassette (also known as pre-cast gels), such as PreciseTM and pre-cast DALT Gel (Amersham ⁇ ) among others, which further includes the staining composition.
  • the staining composition of the kit may be also in a ready-to-use form and in certain cases may be an integral part of the cassette which, upon a single step, begins to adsorb into the gel for detecting the biological moieties separated therein.
  • This method of incoiporation of the dye in the porous substrate is suitable for all kind of dyes having the complexation property with proteins, including, but not limited to, CoomassieTM Blue and Sypro® Ruby.
  • Example 1 Staining compositions
  • the staining compositions of the invention are not limited to a certain staining agent and/or a specific ratio between the staining agent and the other components of the staining solutions.
  • the specific amount of stain that is used depends on the quality of the staining that can be obtained among other conditions.
  • One example of a staining solution is provided in Table 1.
  • the composition of staining solution and beads was prepared by mixing 5Og of BioGel P-10 Gel beads (BioRad, Cat. No. 150-4140) having size distribution of 90-180 microns (wet) Avith 500 ml of a CoomassieTM Blue solution as described in Table 1. The mixture was incubated in room temperature for 10 min allowing absorbance of the dye (staining solution) by the beads. Subsequently, the mixture was rinsed in water and maintained in water either as a suspension or a paste. The beads containing the staining solution will generally leak or bleed the dye only very slowly into the suspension medium.
  • the gel beads were mixed with the Sypro® Ruby and acetic acid solutions and heated and dried in a microwave oven.
  • the gel is covered with the bead paste or the gel is immersed in the bead suspension.
  • the time of the staining can vary. High quality of staining can be obtained in times between 10 minutes and up to many hours. No deterioration in background has been observed. After the staining the gel should be rinsed with water.
  • Example 2 Lactoglobulin bands containing quantities of lmkg; 0.5mkg; 100ng; 50ng; 20ng;
  • Electrophoresis was performed at 150V for 30 min. 3. Duration of staining ⁇ 10 minutes, using a suspension of beads as exemplified above in Example 1.
  • the above described CoomassieTM Blue staining procedure resulted in the image presented in Fig.l .
  • the image is of high quality with sensitivity of at least 5 ng protein and high signal to noise ratio due to the very low background staining.
  • Example 2 An SDS PAGE procedure on the same protein sample used in Example 1 was performed on a 1.5 mm thick PA gradient gel. The gel was stained by the CoomassieTM Blue suspension according to the procedure described hereinabove with the exception that the gel was incubated in the staining suspension overnight (Fig. 2).
  • the resulting quantities of protein in each band are (from left to right): lmlcg, 0.5 nilcg, 100 ng, 50 ng, 20 ng, 5 ng and 10 ng (Fig. 2).
  • the results indicate that the staining at the band which corresponds to 5 ng protein is improved relative to the staining of that band after 10 minutes of incubation in the staining mixture (Fig. 1).
  • Example 5 In this example sensitivity test of detection limit of the method of staining was performed utilizing CoomassieTM Blue bead suspension. For this pmpose a Ing quantity of protein was electrophoretically driven in the gel. Figures 4A and 4B present the result of staining the 1 ng quantity on a spot of lmm in diameter.
  • Figure 5 shows the quality of staining after maintaining the gel for two weeks within the staining suspension. The figure indicates that the selectivity of the staining process between the protein bands and the gel prevents the accumulation of background and results in images having high contrast without any need for de staining.
  • Example 6 BSA bands containing quantities of: 2x20 ⁇ g, 2x1 O ⁇ g, 2x5 ⁇ g, 2x2 ⁇ g and 2x1 ⁇ g were run on an Invitrogen Novex gradient gel.
  • the parameters of the SDS PAGE procedure were as follows: Voltage - 150V Duration- 90min
  • the gel was immersed in the water-based solution of beads for the duration of 30 minutes and rinsed in water.
  • the staining allows for the detection of bands down to 1 microgram without any image processing.

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Abstract

The present invention provides compositions and kits comprising porous substrates that retain staining solutions, wherein the staining solutions comprise one or more staining agents that form visible complexes with proteins or other molecular species of interest in a gel. The invention further provides methods for identifying biological moieties in a sample, using the compositions and kits of the invention, with superior signal to noise ratios that do not require destaining procedures.

Description

COMPOSITIONS FOR ONE STEP COLORIMETRIC ANALYSIS AND
METHODS OF USING SAME
FIELD OF THE INVENTION The present invention relates to compositions, kits and methods for identifying and visualizing biological moieties in a sample.
BACKGROUND OF THE INVENTION
Coloiϊmetric methods are commonly used after electrophoretic separation of proteins or other bioparticles, such as polynucleotides. These methods are an essential step in the process of identifying the content of biological samples. The principle of such colorimetiϊc methods is to provide a visible indication of the species separated along the gel, by using staining agents that selectively form complexes with said species and less selectively form complexes with the surrounding gel. In addition to identifying the content of biological samples, colorimetric methods are also used as an initial purification step.
Numerous staining protocols, staining agents and ready-to-use compositions are commercially available and are generally utilized for colorimetric methods. The most common staining procedures include Coomassie™ Blue, Silver or fluorescent Sypro® Ruby, Ponceau, fluorescamine®, Light Green, Fast Green, Amido Black or a mixture thereof (Romano and Wirth, J. Chromatography, 698: 123-143, 1995).
Commonly used staining procedures often result in excess dye accumulating in the surrounding gel or support matrix as well as forming the desired complexes with the species of interest. As a consequence destaining is required to remove excess dye. US Patent No. 4,219,337 discloses an assay for proteins or polypeptides in solution using Coomassie Brilliant Blue G250 dye in perchloric or hydrochloric acid. Upon mixture of the acidic reagent with a protein sample, the dye couples with the protein and undergoes a color change, following which the absorbance of the mixture can be measured to provide a quantitative analysis of the protein in the sample. US Patent No. 4,839,294 discloses an assay useful for the evaluation of the lipid content of a biological fluid in aqueous dispersion. The assay utilizes a dye-detergent complex which when solubilized by an aqueous dispersion of lipid-containing fluid sample, exhibits a characteristic color. US Patent No. 6,057,160 discloses a method for making Coomassie Brilliant
Blue (CBB) dye/protein complex, comprising mixing a protein with a non-alcoholic aqueous CBB solution having a pH greater than 4, heating the mixture to a first temperature for a short pulse period, then cooling to a second temperature to form a solution containing the CBB dye/protein complex. US Patent No. 6,277,643 discloses a method for the detection of protein on polyacrylamide gels comprising the steps of staining the polyacrylamide gels with a counter-dye composition containing an acidic organic dye and a basic organic dye, selected from the group consisting of Zincon and Ethyl violet, Zincon and Methyl violet, Zincon and Meldola's blue, Coomassie brilliant blue R and Phenosafranin, Coomassie brilliant blue G and Methyl orange, Calconcarboxylic acid and Rhodamine B, and Eriochrome black T and Rhodamine B and protein complexes with the counter- dye composition, and observing protein-counter-dye complexes.
The above mentioned staining procedures as well as the other methods available for staining biological moieties in a gel are tedious, time consuming and usually require several steps including destaining of the gels to reduce the background signal formed by diffusion and accumulation of the staining agents in the gel. This background staining is the limiting factor that determines the detection sensitivity of the colorimetric procedures.
There is an unmet need to a simple, cost-effective, rapid and highly sensitive method for visual identification of biological moieties in a gel.
SUMMARY OF THE INVENTION
The present invention provides compositions, methods and kits comprising porous substrates, including beads, small particles, porous sheets and plates among others and staining compositions comprising one or more staining agents that form visible
_o_ complexes with proteins in a gel. The invention further provides methods for identifying biological moieties in a sample, using the compositions and kits of the invention.
The methods of the invention confer distinct advantages over colorimetric methods known in the art as they provide an exceptionally high signal to noise ratio of stained moieties in a gel. Due to the low background signal (noise), the methods of the invention generally do not require any destaining step as the signals obtained from the protein or other molecular species of interest are clear and can be analyzed or further purified, as is.
The methods of the invention utilize for the first time delivery means that discriminate with exceptional sensitivity between the protein bands incoiporated in the gel and the gel itself. According to this principle, the gel retains almost no staining agent and advantageously no destaining is required. In view of these advantages, the methods of the invention are significantly faster than conventional staining procedures, requiring only one step, which may take no more than several minutes. The one-step method is particularly simple and thus no particular training is required for its application.
According to one aspect, the present invention provides a composition for staining a biological moiety comprising a porous substrate comprising at least one staining component capable of forming a visual signal upon contact with a biological moiety. According to one embodiment, the staining component is absorbed onto the porous substrate. According to another embodiment, the staining component is contained within the porous substrate. The porous substrate is insoluble in the surrounding medium into which the staining agents are released.
The retention or adsorption of the staining agent to the porous substrate is sufficient largely to prevent non specific background staining and yet to permit complex formation with the species of interest thereby increasing the signal to noise ratio.
According to another embodiment the staining component comprises at least one staining agent.
According to another embodiment, the at least one staining agent is selected from the group consisting of: Coomassie™ Blue, Silver stain, Sypro® Ruby, Ponceau, fluorescamine®, Light Green, Fast Green, Amido Black, Ethydium Bromide and Propidium Bromide.
According to yet another embodiment, the staining component comprises a plurality of staining agents. According to yet another embodiment, the porous substrate comprises a polymer, wherein the polymer is linear or cross-linked. According to yet another embodiment, the porous substrate is selected from the group consisting of: porous bead, porous small particles, porous sheets, porous plates and porous fibers.
According to yet another embodiment., the composition is in a form of ready to use suspensions wherein the porous substrate particles are suspended in an appropriate solution. According to yet another embodiment, the composition is in a dry form.
According to yet another embodiment the dry form composition is capable of being transformed into a ready-to-use form upon reconstitution in a carrier. According to this embodiment the carrier is selected from the group consisting of: a hydrophilic solution, a water based-solution, water and a buffer.
According to certain embodiments the compositions of the invention may advantageously be reusable, inasmuch as only a relatively small amount of the total dye contained within the porous substrate is released during each staining procedure.
According to another aspect, the present invention provides a kit comprising a first component comprising a staining component capable of forming a visual signal upon contact with a biological moiety and a second component comprising porous substrate. According to one embodiment, the staining component is absorbed onto the porous substrate. According to another embodiment, the staining component is contained within the porous substrate. According to another embodiment the staining component comprises at least one staining agent. According to another embodiment, the at least one staining agent is selected from the group consisting of: Coomassie™ Blue, Silver stain, Sypro® Ruby, Ponceau, fluorescamine®, Light Green, Fast Green, Amido Black, Ethydium Bromide and Propidium Bromide. According to yet another embodiment, the staining component comprises a plurality of staining agents. According to yet another embodiment, the porous substrate comprises a polymer, wherein the polymer is linear or cross-linked. According to yet another embodiment, the porous substrate is selected from the group consisting of: porous bead, porous small particles, porous sheets, porous plates and porous fibers. According to yet another embodiment, the staining agent is in the form of a ready- to-use composition. According to yet another embodiment, the staining agent is ready to use upon reconstitution in a carrier. According to yet another embodiment, the kit further comprises a carrier. According to yet another embodiment, the carrier is selected from the group consisting of: a hydrophilic solution, a water based-solution, water and a buffer.
According to yet another aspect, the present invention provides a one-step method for staining at least one biological moiety in a gel, the method comprising contacting the gel with a staining composition comprising a staining component comprising at least one staining agent capable of forming a visual signal upon contact with a biological moiety and a porous substrate.
According to one embodiment, the method further comprises the step of incubating the composition with the gel until a visual indication is obtained. According to another embodiment, the method further comprises the step of rinsing the gel with a rinsing solution. According to yet another embodiment, the rinsing solution is selected from a group consisting of: a hydrophilic solution, a water based-solution, water and a buffer.
According to yet another embodiment the contacting step includes immersing the gel in the composition by mechanical means.
According to yet another embodiment the contacting step includes spraying the composition over the gel.
According to yet another aspect, the present invention provides a method for staining at least one biological moiety in a gel, the method comprising: providing a composition comprising a staining component comprising at least one staining agent capable of forming a visual signal upon contact with a biological moiety and at least one porous substrate, wherein the staining component is contained within the at least one porous substrate; providing a gel containing biological moieties; and contacting the gel with the composition .
According to some embodiments the staining composition comprises a plurality of porous substrates. According to some embodiments the staining composition comprises a plurality of staining agents.
According to one embodiment, the method comprises incubating the composition with the gel until a desired visual indication is obtained. According to another embodiment, the method comprises rinsing the gel with a rinsing solution. According to yet another embodiment, the rinsing solution is selected from a group consisting of: a hydrophilic solution, a water based-solution, water and a buffer.
According to yet another embodiment, contacting the gel with the composition is carried out by immersing the gel in the composition. According to yet another embodiment, contacting the gel with the composition is carried out by spraying the composition over the gel. According to yet another embodiment, contacting the gel with the composition is earned out by mechanical means.
According to yet another embodiment, the composition is prepared by a method comprising: providing a staining agent; providing a porous substrate comprising a plurality of porous beads; and mixing the staining agent and the porous beads; and incubating the mixture until at least a portion of the staining agent is adsorbed in the beads; and, optionally, rinsing the mixture with a rinsing solution.
According to one embodiment, the staining agent is prepared prior to being mixed with the porous substrate by solution or reconstitution in a suitable carrier.
According to another embodiment, the porous substrate is selected from but not limited to: porous bead, porous small particles, porous sheets, porous plates and porous fibers. Further embodiments and the full scope of applicability of the present invention will become apparent from the detailed description given hereinafter. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 demonstrates SDS PAGE of protein bands stained by the method of invention on a 0.5 mm acrylamide gel.
Figure 2 presents lactogolobiilin protein bands on a 1.5 mm acrylamide gel.
Figure 3 shows the results obtained from staining protein mass markers (Biorad®) on a 0.5 mm gel.
Figure 4 shows staining obtained from 1 ng of protein of after 8 hr (A) and 15 hr (B) of incubation within the staining mixture.
Figure 5 demonstrates staining of protein spots after two weeks of incubation in the staining composition of the invention.
Figure 6 shows the results obtained from staining BSA bands, following the separation, using the Sypro® Ruby staining agent in a composition of the invention. The result was obtained after 30 minutes.
DETAILED DESCRIPTION OF THE INVENTION
The principle of the staining composition of the present invention is that the staining solution is based on the incorporation of the dye within a porous matrix (e.g. beads, small particles, sheets and fibers). The term 'beads' is used hereinafter to describe any suitable porous material, including sheets, fibers and small particles, which may be used in the context of the present invention. Since the dye is adsorbed in the beads and is held in the porous matrix by capillary forces, the diffusion process by which the dye can leak from the beads is quite slow. Therefore upon adding the composition of the invention to a solution or onto a gel only relatively very small quantities of the total dye escape the porous beads.
The present invention varies from the prior art staining compositions and methods in several respects. First, the present invention is adapted to identify the content of a sample dispersed in a gel by a single step, namely the step of contacting the gel with the staining solution followed by an optional quick and simple rinsing of the gel after a few minutes of contact without the need for a destaining step. Thus, using the compositions and methods of the present invention for protein identification does not require particular skills due to the simplicity of the process. Second, the present invention provides visible indication of the content of a biological sample with significantly high signal to noise ratio. In fact, the background signal is exceptionally low as compared to the background signals obtained with other colorimetric methods for non-specific identification of biological moieties, known in the art. Third, due to the outstanding signal to noise ratio which is obtained within several minutes, the present invention provides an extremely fast procedure for clear identification of various moieties in a gel.
The term "porous substrate" as used herein refers to any porous substrate, such as beads, fibers, sheets and small particles, capable of adsorbing a solution, particularly a solution comprising one or more staining agents. In one embodiment, the rate of diffusion of the staining agent out of the beads, towards an inert environment which is devoid of proteins and/or other biological moieties, is sufficiently slow such that biological moieties (e.g., proteins, nucleic acids, etc.) are preferentially and selectively stained over the inert environment. The porous substrate has the ability to release the dye within a gel containing biological moieties, such as a poly aery lamide gel or an agarose gel. The porous substrate is typically formed from a polymer that serves as a support for some components of a reaction system. In certain embodiments, it may contain an appropriate functional group that can be bound to chemical or biological moieties, e.g., by electrostatic or hydrophobic interactions, or may function as a reagent, catalyst and protecting function, etc. The polymer can be linear or crosslinked. The polymer will be suitable to form beads or other forms of porous substrates that are insoluble macroscopically in the medium to which they are exposed but can swell in an appropriate solvent.
Various porous beads that can be used in the context of the present invention are known in the art and certain beads are also commercially available. Such beads include beads obtained from phenolic complexes of water-insoluble but water-swellable crosslinked N-vinyl-lactam or N-alkyl-N-vinylamide polymers as disclosed in US
Patent No. 3,954,682.
A porous, bead-like copolymer is disclosed in US Patent No. 4,686,243. The bead is obtained by suspension polymerization of monomeric oxiranylalkyl compounds and/or 2-aziridinylalkyl compounds, n-valent crosslinking agents, N-vinylamides, heterocyclic 5-membered ring compounds or 6-membered ring compounds, having a polymerizable olefinic group in each case, and polymerizable quaternary ammonium salts.
A durable and sprayable controlled release bead of active ingredient in the pores of a polymeric micro-porous bead is disclosed US Patent No. 5,888,930. The bead has an anisotropic pore structure of large pores in the interior and small pores at the surface, wherein the gradation of pore sizes between the interior and the surface being continuous.
US Patent No. 5,306,632 discloses a porous shaped substrate, such as a porous bead, having a hydrophilic surface containing amide groups constituting about 1.8 mole percent to less than about 15 mole percent of the total nitrile groups, and containing no amide or carboxyl groups. The substrate is substantially non-swellable in water and is able to resist pressures in a columnar bed of up to about 3000 psi without collapsing. In forming the amide groups, polyacrylonitrile or copolymer thereof containing nitrile groups, an alkaline catalyst such as sodium hydroxide and a nonsolvent for the substrate such as methanol are combined to form a suspension. A peroxide is added to the suspension and the suspension is heated to hydrolyze nitrile groups to amide groups.
Succinylated aminoethyl groups or activated carboxyl groups can be formed on the substrate and a bioactive ligand such as />~aminobenzamidine can be covalently bound to the substrate. The bound ligand can then be used to complex a biologically active substance such as an enzyme to separate the substance from solution. A composition comprising a porous bead and a pigment for cosmetic applications is disclosed in US Patent No. 6,171,602. However, the composition is formulated to prevent bleeding of the pigment out of the bead and for this purpose the composition must contain a hydroalcoholic base. The composition of the present invention is formulated to enable specific bleeding or release of the dye upon contact with biological moieties, but not upon contact with other materials particularly gels.
The porous bead for the composition of the invention may be purchased or prepared by any method known in the art. For example, US Patent No. 4,229,547 discloses a polymerization process which produces spherical beads of polymer having excellent porosity and unusually high bulk density. The process comprises polymerizing discrete droplets of liquid monomer containing a monomer-soluble free radical type catalyst while suspended in aqueous mucilage having plastic flow properties and in the presence of a nonionic surfactant having the proper Hydrophile-Lipophile Balance (HLB), such as, for example, sorbitan monooleate. A method for preparing an improved porous polymer bead is disclosed in US
Patent No. 5,369,133. The method comprises introducing a mixture comprising a purified monomer, initiator and pore-forming agent into a 4-necked reactor with a suspension stabilizer and aqueous solution, suspending the mixture to provide homogeneous organic droplets at a temperature sufficiently low so as not to decompose the initiator, increasing the temperature gradually to a final reaction temperature and maintaining the temperature at the final temperature to react completely by suspension- polymerization the monomers in the individual organic droplet to provide the porous polymer bead, separating the bead, washing the bead with water, acetone and tetrahydrofuran, successively, and drying the bead, precipitating the bead in a solution mixed with monomer and thinner to fill the pores of the porous bead with the diluted monomers, and introducing in a 4-necked reactor with a suspension stabilizer and aqueous solution, and by suspension-polymerization again, to coat the lightly cross- linked gel-type polymer on the surface of pores in the porous polymer bead, and again washing and drying the beads to provide an improved porous polymer bead. The term "biological moiety" as used herein, refers to either man-made or natural proteins, protein-DNA complexes, DNA, RNA, enzymes, peptides, polypeptides, antibodies, antigens, antigenic epitopes and variants thereof, polynucleotides, hormones, carbohydrates, lipids, phospholipids and biotinylated probes.
When the bead comes in contact with the protein band in the gel the dye which diffuses out from the bead is immediately attracted to the proteins in the gel and a concentration gradient is established which facilitates the release of the dye from the polymer beads. This process, also termed herein "complexation", is a pump-like process.
The bead based staining solution can be applied to the gel either by submerging the gel in a water suspension of the beads or by spraying the gel with the suspension of beads or by using a specially designed applicator.
This staining solution can be easily incorporated in any electrophoresis set up such as standard cassette designs among others.
The staining compound can be supplied in the form of a kit comprising two separate components, the dye as the first component and the beads at the second components. The two components may be mixed extemporaneously at the time of use.
The present invention further provides a one-step method for staining at least one biological moiety in a gel, the method comprising contacting the gel with a staining composition comprising a staining component comprising at least one staining agent capable of forming a visual signal upon contact with a biological moiety and a porous substrate.
According to one embodiment, the method further comprises the step of incubating the composition with the gel until a visual indication is obtained. According to another embodiment, the method further comprises the step of rinsing the gel with a rinsing solution. According to yet another embodiment, the rinsing solution is selected from a group consisting of: a hydrophilic solution, a water based-solution, water and a buffer.
According to yet another embodiment the contacting step includes immersing the gel in the composition by mechanical means.
According to yet another embodiment the contacting step includes spraying the composition over the gel. The invention further provides a method for staining biological moiety in a gel, the method comprising: providing a composition comprising a staining component comprising at least one staining agent capable of forming a visual signal upon contact with a biological moiety and porous substrates; providing a gel containing biological moieties; and contacting the composition with the gel.
According to one embodiment, the method comprising incubating the composition with the gel until a desired visual indication is obtained. According to another embodiment, the method comprises rinsing the gel with a rinsing solution. According to yet another embodiment, the rinsing solution is selected from a group consisting of: hydrophilic solution, water based-solution, water and a buffer.
According to yet another embodiment, contacting the composition with the gel is carried out by immersing the gel in the composition. According to yet another embodiment, contacting the composition with the gel is carried out by spraying the composition over the gel. According to yet another embodiment, contacting the composition with the gel is carried out by mechanical means, such as clamps.
According to yet another embodiment, the composition is prepared by a method comprising: providing a staining agent; providing porous substrates; and mixing the staining agent and the porous substrate; and incubating the mixture until at least a portion of the staining agent is adsorbed in the porous substrate; and, optionally, rinsing the mixture with a rinsing solution.
According to one embodiment, the staining agent is prepared prior to being mixed with the porous substrates by reconstitution in a carrier.
The kit of the invention may include a ready-to-use gel cassette (also known as pre-cast gels), such as Precise™ and pre-cast DALT Gel (Amersham©) among others, which further includes the staining composition. The staining composition of the kit may be also in a ready-to-use form and in certain cases may be an integral part of the cassette which, upon a single step, begins to adsorb into the gel for detecting the biological moieties separated therein. This method of incoiporation of the dye in the porous substrate is suitable for all kind of dyes having the complexation property with proteins, including, but not limited to, Coomassie™ Blue and Sypro® Ruby.
The following examples are presented in order to more fully illustrate certain embodiments of the invention. They should in no way, however, be construed as limiting the broad scope of the invention. One skilled in the ait can readily devise many variations and modifications of the principles disclosed herein without departing from the scope of the invention.
EXAMPLES
Example 1 a. Staining compositions
As detailed above, the staining compositions of the invention are not limited to a certain staining agent and/or a specific ratio between the staining agent and the other components of the staining solutions. The specific amount of stain that is used depends on the quality of the staining that can be obtained among other conditions. One example of a staining solution is provided in Table 1.
Table 1. An exemplary composition of a staining solution
Figure imgf000015_0001
The composition of staining solution and beads was prepared by mixing 5Og of BioGel P-10 Gel beads (BioRad, Cat. No. 150-4140) having size distribution of 90-180 microns (wet) Avith 500 ml of a Coomassie™ Blue solution as described in Table 1. The mixture was incubated in room temperature for 10 min allowing absorbance of the dye (staining solution) by the beads. Subsequently, the mixture was rinsed in water and maintained in water either as a suspension or a paste. The beads containing the staining solution will generally leak or bleed the dye only very slowly into the suspension medium.
Svpro® Ruby staining composition
An exemplary composition of a Sypro® Ruby based staining solution is given as follows:
Materials:
15ml Sypro® Ruby protein gel stain (Molecular Probes, Invitrogen detection technologies)
Ig Biogel P-10 Gel beads (Biorad, Cat.N. 150-4140) 3 ml Acetic acid (10%)
The gel beads were mixed with the Sypro® Ruby and acetic acid solutions and heated and dried in a microwave oven.
10ml of distilled deionized water (DDW) was added to the dry mixture to prepare the staining solution.
b. Staining procedure
To perform the staining the gel is covered with the bead paste or the gel is immersed in the bead suspension. The time of the staining can vary. High quality of staining can be obtained in times between 10 minutes and up to many hours. No deterioration in background has been observed. After the staining the gel should be rinsed with water.
Example 2 Lactoglobulin bands containing quantities of lmkg; 0.5mkg; 100ng; 50ng; 20ng;
10ng; 5ng were submitted to SDS PAGE on a 4-20% polyacrylamide gel. The parameters of the SDS PAGE analysis were as follows:
1. Gel thickness - 0.5 mm.
2. Electrophoresis was performed at 150V for 30 min. 3. Duration of staining ~ 10 minutes, using a suspension of beads as exemplified above in Example 1.
The above described Coomassie™ Blue staining procedure resulted in the image presented in Fig.l . The image is of high quality with sensitivity of at least 5 ng protein and high signal to noise ratio due to the very low background staining.
Example 3
An SDS PAGE procedure on the same protein sample used in Example 1 was performed on a 1.5 mm thick PA gradient gel. The gel was stained by the Coomassie™ Blue suspension according to the procedure described hereinabove with the exception that the gel was incubated in the staining suspension overnight (Fig. 2).
The resulting quantities of protein in each band are (from left to right): lmlcg, 0.5 nilcg, 100 ng, 50 ng, 20 ng, 5 ng and 10 ng (Fig. 2). The results indicate that the staining at the band which corresponds to 5 ng protein is improved relative to the staining of that band after 10 minutes of incubation in the staining mixture (Fig. 1).
Example 4
100 ng quantities of Biorad mass markers were run on a 0.5 mm thick gradient gel. The gel was left in the Coomassie™ Blue bead suspension for several days. 10 different mass protein markers are clearly visible in each of the four columns. The result of staining is presented in Fig.3.
Example 5 In this example sensitivity test of detection limit of the method of staining was performed utilizing Coomassie™ Blue bead suspension. For this pmpose a Ing quantity of protein was electrophoretically driven in the gel. Figures 4A and 4B present the result of staining the 1 ng quantity on a spot of lmm in diameter.
Figure 5 shows the quality of staining after maintaining the gel for two weeks within the staining suspension. The figure indicates that the selectivity of the staining process between the protein bands and the gel prevents the accumulation of background and results in images having high contrast without any need for de staining.
Example 6 BSA bands containing quantities of: 2x20 μg, 2x1 Oμg, 2x5μg, 2x2μg and 2x1 μg were run on an Invitrogen Novex gradient gel. The parameters of the SDS PAGE procedure were as follows: Voltage - 150V Duration- 90min
Following the separation the gel was stained by the Sypro® Ruby bead suspension as described above in Example 1
The gel was immersed in the water-based solution of beads for the duration of 30 minutes and rinsed in water.
The resulting image is presented in Fig. 6
The staining allows for the detection of bands down to 1 microgram without any image processing.
The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying current knowledge, readily modify and/or adapt for various applications such specific embodiments without undue experimentation and without departing from the generic concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the puipose of description and not of limitation. The means, materials, and steps for carrying out various disclosed functions may take a variety of alternative forms without departing from the invention.

Claims

1. A composition for staining a biological moiety comprising a porous substrate comprising at least one staining component capable of forming a visual signal upon contact with a biological moiety.
2. The composition of claim 1, wherein the staining component is absorbed onto the porous substrate.
3. The composition of claim 1, wherein the staining component is contained within the porous substrate.
4. The composition of claim 1, wherein the staining component comprises at least one staining agent.
5. The composition of claim 4, wherein the at least one staining agent is selected from the group consisting of: Coomassie™ Blue, Silver stain, Sypro® Ruby, Ponceau, fluorescamine®, Light Green, Fast Green, Amido Black, Ethydium Bromide and Propidium Bromide.
6. The composition of claim 1, wherein the staining component comprises a plurality of staining agents.
7. The composition of claim 1 , wherein the porous substrate comprises a polymer, wherein the polymer is linear or cross-linked.
8. The composition of claim 1, wherein the porous substrate is selected from the group consisting of: porous bead, porous small particles, porous sheets, porous plates and porous fibers.
9. The composition of claim 1 , wherein the composition is in a form of a ready-to- use solution.
10. The composition of claim 1 , in a dry form wherein the composition is capable of being transformed into a ready-to-use form upon reconstitution in a carrier.
11. The composition of claim 10, wherein the carrier is selected from the group consisting of: a hydrophilic solution, a water based-solution, water and a buffer.
12. A kit comprising a first component comprising a staining component capable of forming a visual signal upon contact with a biological moiety; and a second component comprising porous substrate.
13. The kit of claim 12, wherein the staining component is absorbed onto the porous substrate.
14. The kit of claim 12, wherein the staining component is contained within the porous substrate.
15. The composition of claim 12, wherein the staining component comprises at least one staining agent.
16. The kit of claim 15, wherein the at least one staining agent is selected from the group consisting of: Coomassie™ Blue, Silver stain, Sypro® Ruby, Ponceau, fluorescamine®, Light Green, Fast Green and Amido Black.
17. The kit of claim 12, wherein the staining component comprises a plurality of staining agents.
18. The kit of claim 12, wherein the porous substrate comprises a polymer, the polymer is linear or cross-linked.
19. The kit of claim 12, wherein the porous substrates are selected from the group consisting of: porous bead, porous small particles, porous sheets, porous plates and porous fibers.
20. The kit of claim 12, wherein the composition is in a form of a ready-to-use solution.
21. The kit of claim 12, wherein the staining component is in a dry form capable of being transformed into a ready-to-use form upon reconstitution in a carrier.
22. The kit of claim 12, further comprising a earner for reconstituting the staining component.
23. The kit of claim 22, wherein the carrier is selected from the group consisting of: a hydrophilic solution, a water based-solution, water and a buffer.
24. A one-step method for staining at least one biological moiety in a gel, the method comprising contacting the gel with a staining composition comprising a porous substrate comprising at least one staining component capable of forming a visual signal upon contact with a biological moiety.
25. The method of claim 24, further comprising the step of incubating the composition with the gel until a visual indication is obtained.
26. The method of claim 24, further comprising the step of rinsing the gel with a rinsing solution.
27. The methods of claim 26, wherein the rinsing solution is selected from a group consisting of: a hydrophilic solution, a water based-solution, water and a buffer.
28. The method of claim 24, wherein the contacting step includes immersing the gel in the composition or spreading the composition on the gel using mechanical means.
29. The method of claim 24, wherein the contacting step includes spraying the composition over the gel.
30. The method of claim 24, wherein the porous substrate comprises a polymer, the polymer is linear or cross-linked.
31. The method of claim 24, wherein the staining component comprises at least one staining agent.
32. The method of claim 31, wherein the at least one staining agent is selected from the group consisting of: Coomassie™ Blue, Silver stain, Sypro® Ruby, Ponceau, fluorescaniine®, Light Green, Fast Green, Amido Black, Ethydium
Bromide and Propidium Bromide.
33. The method of claim 24, wherein the staining component comprises a plurality of staining agents.
34. The method of claim 24, wherein the porous substrate is selected from the group consisting of: porous bead, porous small particles, porous sheets, porous plates and porous fibers.
35. A method for staining a biological moiety in a gel, the method comprising: a. providing a staining composition comprising a porous substrate comprising at least one staining component capable of forming a visual signal upon contact with a biological moiety, b. providing a gel containing at least one biological moiety; and c. contacting the gel with the staining composition.
36. The method of claim 35, wherein the staining composition is prepared by: providing at least one staining component capable of forming a visual signal upon contact with a biological moiety; providing porous substrate; mixing the staining component and the porous substrate to form a mixture; incubating the mixture until at least a portion of the staining agent is adsorbed onto porous substrates; and, optionally, rinsing the mixture with a rinsing solution.
37. The method of claim 35, wherein the staining component comprises at least one staining agent.
38. The method of claim 37, wherein the at least one staining agent is selected from the group consisting of: Coomassie™ Blue, Silver stain, Sypro® Ruby, Ponceau, fluorescamine®, Light Green, Fast Green, Amido Black, Ethydium Bromide and Propidium Bromide.
39. The method of claim 37, wherein the staining agent is in a form of a ready-to- use solution.
40. The method of claim 37, wherein the staining agent is in a dry form wherein the composition is capable of being transformed into a ready-to-use form upon reconstitution in a carrier.
41. The method of claim 40, wherein the carrier is selected from the group consisting of: a hydrophilic solution, a water based-solution, water and a buffer.
42. The method of claim 35, wherein the rinsing solution is selected from the group consisting of: a hydrophilic solution, a water based-solution, water and a buffer.
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Citations (3)

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Publication number Priority date Publication date Assignee Title
US5776684A (en) * 1995-12-28 1998-07-07 Edvotek Method for staining biomolecules using a gelled matrix
WO1999042620A2 (en) * 1998-02-19 1999-08-26 Edvotek Articles of manufacture and methods for staining biomolecules
US20010044108A1 (en) * 2000-01-28 2001-11-22 Lih-Bin Shih Detection of biomolecules in gels following electrophoresis

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Publication number Priority date Publication date Assignee Title
US5776684A (en) * 1995-12-28 1998-07-07 Edvotek Method for staining biomolecules using a gelled matrix
WO1999042620A2 (en) * 1998-02-19 1999-08-26 Edvotek Articles of manufacture and methods for staining biomolecules
US20010044108A1 (en) * 2000-01-28 2001-11-22 Lih-Bin Shih Detection of biomolecules in gels following electrophoresis

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