WO2008092181A2

WO2008092181A2 - Method for analysing electrophoresis gels

Info

Publication number: WO2008092181A2
Application number: PCT/AT2008/000032
Authority: WO
Inventors: Christian Reichel; Igor HOLLÄNDER; Irene Lichtscheidl; Hans Kroath
Original assignee: Austrian Research Centers Gmbh - Arc
Priority date: 2007-01-31
Filing date: 2008-01-31
Publication date: 2008-08-07
Also published as: WO2008092181A3; AT504849B1; AT504849A1

Abstract

The present invention relates to a method for visualizing at least one protein band and/or at least one protein spot'on an at least two-dimensional electrophoresis gel comprising the steps: providing an at least two-dimensional electrophoresis gel comprising said at least one protein band and/or said at least one protein spot; visualizing said at least one protein band and/or said at least one protein spot using a three-dimensional imaging technique, such as confocal microscopy.

Description

Methods for analysing electrophoresis gels

The present invention relates to a method for visualizing electrophoresis gels.

Gel electrophoresis is frequently used in molecular biology, genetics, microbiology and biochemistry for separating nucleic acids, proteins, glycoproteins, carbohydrates etc.. The results can be analysed quantitatively by visualizing the bands and spots on the gel. If the bands and spots on the gel are quantitatively or semi-quantitatively analysed an image is recorded with an appropriate computer operated imaging device (e.g. scanner, camera, etc.), and the intensity of the band or spot of interest is measured and compared against standards or markers loaded on the same gel.

A specific form of gel electrophoresis, which is commonly used to analyse proteins, is two-dimensional gel electrophoresis (2-DE or 2-D electrophoresis) . 2-DE utilizes the fact that proteins vary not only in size but also independently from the size in the isoelectric point. In one dimensional electrophoresis (1- DE or 1-D electrophoresis) , molecules such as proteins are separated in one dimension, so that all the molecules will lie along a line separated from each other by some property (e.g. size) . 2-DE begins with a one dimensional gel electrophoresis but then separates the molecules by a second property in a direction 90° from the first. The result is that the molecules are spread out across a 2-D surface rather than along a line. Because it is less likely that two molecules will be the same in both properties than that they will be the same in just one property, molecules are more effectively separated in 2-DE than in one dimensional electrophoresis. Proteins, for instance, can be separated by this technique according to the isoelectric point and molecular mass (other separation parameters are also possible) .

To separate proteins by isoelectric point, a gradient of pH is applied to a gel and an electric potential is applied across the gel, making one end more positive than the other. At all pHs other than their isoelectric point, proteins will be charged. If they are positively charged, they will be pulled towards the more negative end of the gel and if they are negatively charged they will be pulled to the more positive end of the gel. The _ 9 — proteins applied in the first dimension will move along the gel and will accumulate at their isoelectric point.

Before separating the proteins by mass, they may be treated with detergents like sodium dodecyl sulfate (SDS) . This denatures the proteins and attaches a number of SDS molecules roughly proportional to the protein's length. Because a protein's length (when unfolded) is roughly proportional to its mass, this is equivalent to saying that it attaches a number of SDS molecules roughly proportional to the protein's mass. Since the SDS molecules are negatively charged, the result of this is that all of the proteins will have approximately the same mass- to-charge ratio. In addition, proteins will not migrate when they have no charge (a result of the isoelectric focusing step) therefore the coating of the protein in SDS allows migration of the proteins in the second dimension) . In the second dimension, an electric potential is again applied, but at a 90 degree angle from the first field. The proteins will be attracted to the more positive side of the gel proportionally to their mass-to-charge ratio. As previously explained, this ratio will be nearly the same for all proteins. The proteins' movement will be slowed by frictional forces. The gel therefore acts like a molecular sieve when the voltage is applied, separating the proteins on the basis of their molecular weight with larger proteins being retained higher in the gel and smaller proteins being able to pass through the sieve faster.

The result of this is a gel with proteins spread out within its volume. These proteins can then be visualized by a variety of means, in particular by using stains such as silver, coomassie, fluorescent dyes, radioactivity or another marker detectable by a corresponding imaging method. The intensity of the staining may be used as a quantitative indicator.

The analysis of such 2-DE gels is usually performed with 2- DE gel analysis software using images obtained by conventional scanners or cameras. These tools are well suited for quantification and analysis of well-defined well-separated molecule spots and bands (e.g. protein spots on 2-DE gels) . However, they deliver wrong, not reproducible and not evaluable results with less-defined, less-separated spots and bands. This problem, however, may also occur with one dimensional electrophoresis gels . Therefore, it is an object of the present invention to provide methods which allow analysing incompletely separated (overlapping) spots and bands (less-defined and/or separated) , weak spots and bands ("noise"; e.g., "ghost spots").

This object is achieved by the present invention which provides a method which allows to visualise three-dimensional bands and spots on an electrophoresis gel by using a three-dimensional imaging technique.

Therefore, the present invention relates to a method for visualizing at least one protein band and/or at least one protein spot on an at least two-dimensional electrophoresis gel comprising the steps:

- providing an at least two-dimensional electrophoresis gel comprising said at least one protein band and/or said at least one protein spot,

- visualizing said at least one protein band and/or said at least one protein spot using a three-dimensional imaging technique.

Conventional methods for visualising protein bands and spots on electrophoresis gels use simple images obtained by scanners and cameras. This type of imaging allows only a two-dimensional visualisation of bands and spots on electrophoresis gels. -Modern software packages and imaging tools, however, include advanced features, which simulate a three-dimensional image of an electrophoresis gel without physically scanning the gel in its third dimension. These "three-dimensional" images are obtained from two-dimensional images, the third dimension reflecting the intensity of the bands and spots on the electrophoresis gel. The intensity of the bands and spots on the electrophoresis gel correlates with the amount of molecules present in said bands and spots. Therefore these images cannot be considered as being three-dimensional images of the electrophoresis gel and they do not allow detecting and distinguishing, for instance, overlapping spots or bands. In contrast thereto, the method according to the present invention allows to visualise protein bands and spots on an electrophoresis gel which partly or completely overlap. With conventional methods overlapping protein bands and spots are only detected as one single band or spot or as an undefined smear.

Methods and systems (apparatuses) to obtain three-dimension- al images of substantially transparent three-dimensional objects are known to the person skilled in the art and are disclosed, for instance in the US 5,804,813, T. Furukawa (Editor): Biological Imaging and Sensing. Springer; 2006, ISBN-13: 978-3540438984; P. Michael Conn (Editor) : Imaging in Biological Research, Part A, Volume 385 (Methods in Enzymology) , Academic Press, 2004, ISBN-13: 978-0121827908.

The method according to the present invention is suited to analyse electrophoresis gels which are obtained by separating proteins of an inhomogeneous source (e.g. cell extracts, body fluids and tissues) or purified proteins (e.g. glycoproteins).

In the art it is known to three-dimensionally visualise nucleic acid molecules separated by electrophoresis gels using con- focal fluorescence microscopy (Baert P et al. Cytometry A (2003) 51A.-26-34). The nucleic acid molecules analysed therein can only be visualised in the electrophoresis gel because they form single spots/bands of the same kind of nucleic acid molecule. Hence, the visualisation of samples comprising a high variation of molecules on an electrophoresis gel using confocal microscopy - as it is the case when proteins from an inhomogeneous source are separated in gel electrophoresis - was according to Baert P et al. not regarded as being achievable. It surprisingly turned out that such visualisation methods which have been applied for (one dimensional or even capillary) DNA gel electrophoresis (wherein detection of overlapping peaks was regarded as not distinguishable) are suitable for the protein gel electrophoresis method according to the present invention - even for the detection of overlapping and similar protein bands.

The term "protein", as used herein, refers to molecules made of amino acids arranged in a linear chain and joined together by peptide bonds between the carboxyl and amino groups of adjacent amino acid residues. According to the present invention a protein comprises preferably at least 10 amino acid residues. The term "protein" within the scope of the present invention may be used interchangeable with the terms "peptide" and "polypeptide".

As used herein, the term "gel" refers to a matrix used to separate molecules. The gel may be a crosslinked polymer whose composition and porosity is chosen based on the weight and composition of the target of the analysis. When separating proteins, polypeptides or peptides the gel may be made with differ- ent concentrations of acrylamide and a cross-linker, producing differently sized mesh networks of polyacrylamide . The gel forms a solid but porous matrix.

The term "electrophoresis", as used herein, refers to the electromotive force (EMF) that is used to push or pull molecules through a matrix, preferably through a gel matrix. The molecules, preferably present in a sample, are put into wells/slots on the gel whereinafter, when an electric current is applied, the molecules will enter the gel matrix and move through the matrix at different rates, towards the anode if negatively charged or towards the cathode if positively charged.

As used herein, the term "at least two-dimensional electrophoresis gel" refers to an electrophoresis gel which is obtainable by methods, wherein a mixture of more than one protein is separated by at least two physical properties in at least two dimensions. Consequently, this term includes also three-, four-, five-, six- or even ten dimensional electrophoresis gels. It is of course possible to separate said proteins in more than one dimension by exploiting one or two physical properties of said proteins (e.g. three-dimensional electrophoresis: first dimension - separation of the proteins according to the isoelectric point, second dimension - separation of the proteins according to mass, third dimension - a further separation of the proteins according to the isoelectric point) . It is understood, that an e . g. four-dimensional electrophoresis is a four-stage process, where four different electrophysical conditions are applied sequentially to a single gel. In case of an one-, two-, or three- dimensional eletrophoresis, these processes usually run in up to three different spatial dimensions; in case of a more-than-three dimensional electrophoresis, more processes share the same spatial dimension or a combination thereof. However, two-dimensional electrophoresis is preferred.

Two-dimensional electrophoresis begins with a first dimensional electrophoresis but then separates the molecules by a second property in a direction preferably 90 degrees from the first. In the first dimensional electrophoresis, proteins (or other molecules) are separated in one dimension, so that all the proteins/molecules will lie along a lane but be separated from each other by a property (e.g. isoelectric point) . The result is that the molecules are spread out across a two-dimensional gel. The two dimensions that proteins are separated into using this technique are isoelectric point and mass. To separate the proteins by isoelectric point, a gradient of pH is applied to a gel and an electric potential is applied across the gel, making one end more positive than the other. At all pHs other than their isoelectric point, proteins will be charged. If they are positively charged, they will be pulled towards the more negative end of the gel and if they are negatively charged they will be pulled to the more positive end of the gel. The proteins applied in the first dimension will move along the gel and will accumulate at their isoelectric point (point at which the overall charge on the protein is 0 (i.e. a neutral charge)). Before separating proteins by mass, they are preferably treated with sodium dodecyl sulfate (SDS) along with other reagents. In the second dimension, an electric potential is again applied, but at a 90 degree angle from the first field. The proteins will be attracted to the more positive side of the gel proportionally to their mass-to-charge ratio. As previously explained, this ratio will be nearly the same for all proteins. The proteins' progress will be slowed by frictional forces. The gel therefore acts like a molecular sieve when the current is applied, separating the proteins on the basis of their molecular weight with larger proteins being retained higher in the gel and smaller proteins being able to pass through the sieve and reach lower regions of the gel.

According to a preferred embodiment of the present invention the three-dimensional imaging technique is based on confocal microscopy, tomography and microtomography .

It turned out that particularly confocal microscopy can be used to three-dimensionally visualize bands and spots on electrophoresis gels. The major optical difference between a conventional microscope and a confocal microscope is the presence of confocal pinholes, which allow only for light from the plane of focus to reach the detector. This forms the principle of a confocal microscope where "out of focus" light is removed from the image by the use of a suitably positioned pinhole. This produces images allowing for the collection of optical slices of the target for use in creating a three-dimensional image of the sample. If the plane of focus is changed or the object moved, a series of images at different positions can be produced through the thickness of the object, i.e. a series of X-Y images at different Z positions. Such a series of images is a three dimensional representation of the target (e.g. electrophoresis gel) produced by optical (as opposed to physical) sectioning.

Confocal microscopes possess several advantages over conventional microscopy. First, confocal microscopy produces images of improved resolution by eliminating out-of-focus light. Confocal microscopes also have a higher level of sensitivity compared to conventional microscopes, due to highly sensitive light detectors and the ability to accumulate images captured over time. Another key advantage of confocal microscopy is the ability to produce three-dimensional images of specimens as mentioned above. Confocal microscopy is also a less invasive form of imaging. This is due to the use of, e.g., high-power laser illumination and the reduction in light-scattering artifacts, allowing the non-invasive imaging of even thick sections provided that these sections are substantially semi-transparent targets.

In confocal microscopy, as in conventional microscopy, it is necessary for the tissue specimen of interest to demonstrate some form of optical contrast between different areas of the sample for visualization. This usually requires staining of the target using a label that either absorbs or reflects light or is fluorescent .

According to a preferred embodiment of the present invention said at least one protein band and/or protein spot is labelled with at least one fluorophore, chromophore, chemiluminescent and/or radiolabel.

In order to visualise the spots and bands on an electrophoresis gel the molecules are labelled before or after the separation on the gel by a chromophore, fluorophore, radioactive marker or any suitable marker corresponding to the imaging method used (e.g. Coomassie, Sypro Ruby, Cy-dyes, 35S, etc.)

The at least one band and/or spot is labelled preferably with a fluorophore selected from the group consisting of acrid- ine orange, Cy3, Cy5, 4 ' , 6-diamidino-2-phenylindole, ethidium bromide, fluorescein, Hilyte fluor, rhodamine, SYBR Green I, SY- BR Green II, SYBR Gold, oxazole yellow, thiazole orange, Pico- Green, Texas Red, Cy2, Sypro Ruby, Sypro Orange, Deep Purple and combinations thereof.

The molecules to be visualized in the gel may be labelled with one or more fluorophores . The labelling of the bands and/or spots with at least two fluorophores allows to distinguish between at least two samples labelled prior its electrophoretic separation (differential analysis) . Therefore the samples may be prior their electrophoretic separation singly or multiply labelled.

Furthermore the use of more than one marker allows to clearly distinguish between similar or even identical proteins derived from more than one source and being part of a single protein spot or band.

According to a further preferred embodiment of the present invention the electrophoresis gel is a polyacrylamide gel or an agarose gel.

The method of the present invention may be performed with any polymer used to manufacture an electrophoresis gel, it being particularly preferred to use polymers which are used to analyse proteins, including polypeptides, peptides and modifications thereof (e.g. glycoproteins).

Proteins (including polypeptides, peptides, and modifications thereof) are usually analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) , by native gel electrophoresis, by quantitative preparative native continuous polyacrylamide gel electrophoresis (QPNC-PAGE) , isoelectric focusing (in polyacrylamide or agarose gels; carrier ampholyte based, immobilized pH-gradient based) or by 2-D electrophoresis.

According to a preferred embodiment of the present invention confocal microscopy involves the use of a confocal laser scanning microscope or a two-photon confocal microscope.

It is particularly advantageous to combine the confocal principle with a scanning system preferably utilizing a laser light source. This builds up an image by scanning a point of laser light across the sample in X and Y directions. In the case of a laser scanning system, the detector of reflected light from the sample is generally a photomultiplier tube. The signal from the photomultiplier tube is converted to a digital form that contains information on the position of the laser in the image and the amount of light coming from the sample. Usually a computer is used to store the intensity value of each point from the detector, and presents these in the correct order on a high- resolution video monitor to display the image. To collect a series of images, the computer then shifts the focus by a fixed amount and the object is scanned again to produce the next image at the different Z position. This image is stored and the process repeated to build the three-dimensional data set.

In a typical set-up for a laser scanning confocal microscope, a pinhole is placed in front of the light source to produce a distinct and spatially constrained illumination point. The light passing through this pinhole is focused on the sample, while a second pinhole is placed in front of the light detector. If the optical distance from the detector pinhole to the focal point is exactly the same as that between the focal point and the illumination pinhole, only the light generated at the focal point will reach the detector since the pinhole will block out the out-of-focus light. The signal from the detector is then digitized and passed preferably to a computer. The image of the sample is digitally built up by scanning the sample in the X and Y directions and then special software is used to reconstruct a digital image.

Two-photon (or multi-photon) microscopy is virtually the same as laser scanning confocal microscopy, except that the need for pinholes is eliminated. Optical sectioning of the sample is instead achieved through the preferred use of a mode-locked Ti: sapphire laser which operates in the near-infrared. The laser produces a high photon density that is tuned to a wavelength about twice that of the intended absorption wavelength of the sample. This means that two or more photons are required at a single point to produce an optical signal (i.e. excitation) that can be detected. The probability of such a two-photon event occurring is limited to the focal plane where there is an extremely high photon density. As a result, in 2-photon imaging, excitation occurs only at the plane of focus.

The use of two-photon confocal microscopy rather than laser scanning confocal microscopy reduced focus bleaching (the loss of optical signal production due to sample damage from the laser) . Two-photon microscopy also increases sample penetration because of the reduced absorption of near-infrared radiation which on the other hand causes little damage to the sample. In addition, two-photon microscopy may increase the sensitivity because the elimination of the pinhole allows the entire signal to reach the detector of the microscope. In some instances it is advantageous to quantify single spots and bands present on an electrophoresis gel. Therefore, the amount of the at least one spot and/or at least one band is quantified.

The use of three-dimensional imaging techniques, in particular confocal microscopy, in the method according to the present invention not only allows analysing the bands and spots on an electrophoresis gel qualitatively but also quantifying the amount of molecules present in a single spot or band. Due to the possibility to separate molecules and to concentrate them in a spot or band by electrophoretic methods, thus obtaining spots and bands containing ideally only one type of molecules, it is possible to quantify these spots and bands.

To quantify the amount of molecules (e.g. proteins) deposited in a spot, another sample with known amount of molecules has to undergo the identical electrophoresis process, preferably within the same gel. Quantification is based on comparison of the amount deposited in a known standard spot with the amount deposited in a spot in question. In this method, the amount is defined as the volume occupied by the spot. The volume is in turn defined as the number of voxels (elementary volume units in a 3D imaging method) representing the given spot such that the characteristic value (intensity) of the voxel is above some defined threshold. The threshold intensity represents the discrimination limit between spot and background, the intensity of the spot voxels being typically substantially higher than the intensity of the background voxels. Since the threshold intensity is uniform for all objects (spots) over the gel, a volumetric comparison between different objects (spots) is possible.

To calculate the volume of a spot, one first specifies the location of the spot, typically by identifying a core voxel located in the middle of the spot mass. This voxel is assumed to have its intensity above the threshold and is therefore declared to belong to the spot. One then identifies all voxels which are spatial neighbours of the core voxel and have their intensities above the threshold. All these voxels are declared to belong to the spot. In a similar way, one then continues to add voxels to the spot, if they are spatial neighbours of the voxels already declared to belong to the spot and if their intensity is above the threshold. The process continues until there are no other voxels which could be potentially added. The total amount of voxels declared to belong to the spot in this way represents the volume of the spot in question. Methods for calculating the volume of protein spots and bands are known to the person skilled in the art.

According to a preferred embodiment of the present invention the at least one protein band and/or the at least one protein spot comprises at least one protein having at least one post- translational modification, preferably selected from the group consisting of acylation, glycosylation, biotinylation, pegyla- tion, alkylation, pyroglutamate formation, phosphorylation, sulfation, oxidation, nitrosylation and deamidation.

The method of the present invention is specifically suited to visualise and analyse proteins which have been posttransla- tionally modified and for analysing mixtures of proteins which have been differently modified posttranslationally, i.e. proteins which have identical primary amino acid sequence but different carbohydrate modification (or other posttranslational modifications, such as acetylation, formylation, γ-carboxyla- tion, etc.). The method according to the present invention is specifically useful to analyse and distinguish such similar protein forms in gel electrophoresis. By using markers/labels which are able to bind specifically to proteins having a distinct kind of posttranslational modification it is possible to label such proteins and to distinguish them on a three-dimensional image obtained with the method of the present invention from proteins which do not have such modification but have similar physical properties. With conventional methods only one single band/spot or a smear may be observed on an electrophoresis gel.

According to another preferred embodiment of the present invention the electrophoresis gel has a thickness of at least 1.5 mm, preferably of at least 4 mm.

It turned out that even better results may be obtained with the method according to the present invention, when the proteins are not applied in the vicinity of the surface of the electrophoresis gel and/or in the vicinity of the interface between electrophoresis gel and its solid support. Therefore, the at least one protein band and/or said at least one protein spot in the electrophoresis gel is preferably located at least 10% (preferably at least 20%, more preferably at least 30%) distant from the upper and/or at least 10% (preferably at least 20%, more preferably at least 30%) distant from the lower side of the electrophoresis gel, wherein said distance is calculated from the overall thickness of the electrophoresis gel.

This may be achieved, e.g. by applying the sample comprising a plurality of proteins in at least one slot of the electrophoresis gel, wherein the bottom of said slot is positioned at least 10% distant from the lower side of the electrophoresis gel. Practically, instead of the 0.5-1.5 mm thickness of the typical gels, thicknesses of 1.5-4 mm are applied according to preferred embodiments of the invention. Moreover, the slots for the protein sample to be analysed are located with suitable distances from the lower surface and filled only to a certain extent, so that at least 10%, preferably at least 20%, especially at least 30% of gel thickness is present below the slot bottom as well as between the upper surface of the sample solution in the slot and the surface of the gel outside the slot so that the sample proteins have a higher distance from the surfaces of the gel. It is understood that the electric field is applied in the direction perpendicular to the said lower and upper surfaces.

Another aspect of the present invention relates to the use of a confocal microscope for the method according to the present invention .

Another aspect of the present invention relates to a three- dimensional presentation of an electrophoresis gel obtainable by a method according to the present invention.

The results of the method can be typically presented in form of perspective drawings/images which reflect the spatial depth of the individual spots. A presentation using software tools for three-dimensional presentation (allowing for interactive manipulation of the visualized objects such as spatial rotation, zooming etc) is also possible. For the purpose of quantification, these drawings have to be accompanied by numerical data representing the volumes of the individual spots. For the experiments presented in this description, standard universal mathematical software allowing computation and visualization of three-dimensional numerical data were used.

A kit based on this method may be an instrument combining the relevant parts of a three-dimensional scanning device (preferably a laser scanning confocal microscope) and a computer running a dedicated software. This software will use standard algorithms but will be customized to the data formats and presentation modes required, and will allow simple user interaction. Typically, the user places the electrophoretic gel on a sample holder plate and starts the software; the user will be presented a preview image of the whole gel; the user will interactively select the region of interest on the gel; the system will do the three-dimensional scanning of the region; the system will present a prespective view of the region of interest with automatically identified spots and their volumes computed. This instrument will therefore be an alternative to the two-dimensional gel documentation systems currently available.

The present invention is further illustrated by the following figures and examples, however, without being restricted thereto.

Fig. Ia shows a side view (Z- and X-axis) of two spots on an 2-DE gel;

Fig. Ib shows a top view (Y- and X-axis) of the same spots as in Fig. Ia. The threshold, as used herein, is the discrimination limit between spot and background. Voxels with intensities below the threshold belong to the background. Voxels with intensities above the threshold are spatially grouped and form the objects/spots. Typically, the higher this threshold, the higher the specificity of the method, but the smaller its sensitivity.

Fig. 2a shows a further side view (Z- and Y-axis) of the spots of Fig. 1;

Fig. 2b shows a three-dimensional view (X-, Y- and Z-axis) of the spots of Fig. 1.

Fig. 3a shows a side view (Z- and X-axis) of some spots on an 2-DE gel;

Fig. 3b shows a top view (Y- and X-axis) of the same spots as in Fig. 3a.

Fig. 4a shows a further side view (Z- and Y-axis) of the spots of Fig. 3;

Fig. 4b shows a three-dimensional view (X-, Y- and Z-axis) of the spots of Fig. 3.

Fig. 5a shows a side view (Z- and X-axis) of some spots on an 2-DE gel;

Fig. 5b shows a top view (Y- and X-axis) of the same spots as in Fig. 5a. Fig. 6a shows a further side view (Z- and Y-axis) of the spots of Fig. 5;

Fig. 6b shows a three-dimensional view (X-, Y- and Z-axis) of the spots of Fig. 5.

Fig. 7a shows a 2-DE gel of an E.coli lysate stained with Sypro ruby. The gel thickness is approx. 1 mm.

Fig. 7b shows an enlarged detail of the gel shown in Fig. 7a.

Fig. 8a shows a two-dimensional image of the detail shown in Fig. 7b recorded with a confocal microscope.

Fig. 8b shows a three-dimensional image of the detail shown in Fig. 8a.

Figs. 9a to 9c show three-dimensional images of spot A as marked in Figs. 7b to 8b, whereby the concentration of the lysate applied was varied from 50 to 200μg.

Figs. 10a to 10c show the quantification of spot A, when various amounts of E.coli lysate were applied on the electrophoresis gels (50 to 200μg) .

EXAMPLES : The method is implemented in the following steps:

1. A gel after electrophoresis and labelling is placed on a desk, and, if necessary, illuminated by a suitable light source (typically, UV) to make the spots visible for the human experimenter.

2. The relevant part of the gel including the spots to be analyzed is cut out of the gel (typically a piece ca . 6 x 10 mm, depending on the size of the sample holder) and placed onto a sample holder. Typically, the sample holder is a microscope slide with a chamber for liquid buffer. Liquid buffer is added to prevent drying out of the gel.

3. Sample holder is placed into the scanning area of the con- focal microsope.

4. Suitable scanning parameters are set, such as objective selection, pinhole size selection, appropriate focusing, frame and line averaging etc. The following parameters (only the most relevant listed) have been set in the experiments by the authors: a. Objective Magnification: 2.5x b. Pinhole Size: 2.19 airy c. Real Volume Size: 6.00 x 6.00 x 1.88 mm³ d. Data Volume: 512 x 512 x 43 voxels e. Voxel Size: 11.72 x 11.72 x 44.76 mm³ f . Line Averaging: 4 x g. Frame Averaging: 4 x

5. Preview mode is used to check the proper settings. Settings are adapted if necessary.

6. Scanning process is started. This can take up to one hour to complete, depending on the parameters.

7. Image data stored by the confocal microscope on the PC are read in by a mathematical software package for three-dimensional data processing. Data format conversions are performed if necessary.

8. Suitable three-dimensional perspective visualization modes are set and the three-dimensional data are graphically presented on the screen as a preview.

9. Based on the preview, a suitable three-dimensional subre- gion and a threshold value are selected.

10. The spatial position of the relevant spots is identified on the preview.

11. The subregion is visualized in a perspective view, featuring the relevant spots as solid objects with surfaces defined by the threshold value. The number of voxels belonging to the respective spots are shown.

12.If a standard spot is available on the same gel with a known amount, the same procedure is performed for this spot. The ratio of the number of voxels for the spot in question and the standard spot can be used to specify the amount of molecules in the spot in question.

Claims

Claims :

1. Method for visualizing at least one protein band and/or at least one protein spot on an at least two-dimensional electrophoresis gel comprising the steps:

2. Method according to claim 1, characterised in that the three- dimensional imaging technique is confocal microscopy, tomography and microtomography.

3. Method according to claim 1 or 2, characterised in that said at least one protein band and/or protein spot is labelled with at least one fluorophore, chromophore, chemiluminescent and/or radiolabel .

4. Method according to claim 3, characterised in that the fluorophore is selected from the group consisting of acridine orange, Cy3, Cy5, 4 ' , 6-diamidino-2-phenylindole, ethidium bromide, fluorescein, Hilyte fluor, rhodamine, SYBR Green I, SYBR Green II, SYBR Gold, oxazole yellow, thiazole orange, PicoGreen, Texas Red, Cy2, Sypro Ruby, Sypro Orange, Deep Purple and combinations thereof.

5. Method according to any one of claims 1 to 4, characterised in that the electrophoresis gel is a polyacrylamide gel or an agarose gel.

6. Method according to any one of claims 2 to 5, characterised in that confocal microscopy involves the use of a confocal laser scanning microscope, a two-photon confocal microcope, or a programmable array microscope.

7. Method according to any one of claims 1 to 6, characterised in that at least one protein spot and/or at least one protein band is quantified.

8. Method according to any one of claims 1 to 7, characterised in that the at least one protein band and/or the at least one protein spot comprises at least one protein having at least one post-translational modification, preferably selected from the group consisting of acylation, glycosylation, biotinylation, pegylation, alkylation, pyroglutamate formation, phosphorylation, sulfation, oxidation, nitrosylation and deamidation.

9. Method according to any one of claims 1 to 8, characterised in that the electrophoresis gel has a thickness of at least 1.5 mm, preferably of at least 4 mm.

10. Method according to any one of claims 1 to 9, characterised in that the at least one protein band and/or said at least one protein spot in the electrophoresis gel is located at least 10% distant from the upper and/or at least 10% distant from the lower side of the electrophoresis gel, wherein said distance is calculated from the overall thickness of the electrophoresis gel .

11. Use of a confocal microscope for the method according to any one of claims 1 to 10.

12. Three-dimensional presentation of an electrophoresis gel obtainable by the method according to any one of claims 1 to 10.