Two-dimensional gel electrophoresis

The two-dimensional gel electrophoresis or 2D gel electrophoresis is an analytical method in biochemistry, molecular biology and proteomics. It was developed in 1975 independently by O'Farrell and Klose. Combining the isoelectric focusing (IEF) with SDS -polyacrylamide gel electrophoresis (SDS-PAGE ) for separating complex mixtures of proteins ( bacterial lysates, the lysates from eukaryotic cells or tissues, body fluids ) in individual proteins. The combination of the two separation techniques orthogonal to each other running a very high resolution separation is achieved.

Each spot (spot) in the protein pattern corresponds to a type ( species) of protein molecules. Since the protein pattern change environment and state-dependent in biological systems, they can be used to distinguish damaged and healthy or optimal and suboptimal grown cells. For example, enter information about the causes of disease or the mechanism of action of drugs at the molecular level. Due to the complexity of two-dimensional protein patterns is resorted to their use on specially developed computer programs.

  • 6.1 digitize 2D gels 6.1.1 Imaging Devices
  • 6.1.2 Single Channel techniques
  • 6.1.3 multiplexing
  • 6.4.1 Referenzgele and proteome
  • Detect and quantify protein spots 6.4.2
  • 6.4.3 visualize data
  • 7.1 Chemistry of the separation system
  • 7.2 Geometry of the separation system

Sample Preparation

The extraction and processing of the sample under identical conditions as possible. This precludes that falsifying influences acting on the specimen to be examined in addition to the experimental variables. Made of extra- cellular compartments ( secreted proteins ), proteins are usually like. Intracellular proteins are extracted by gentle disruption of the cell structures and a partial protein purification. Outside their natural environment, the proteins are particularly prone to the formation of protein aggregates and degradation by proteases. Is also being worked during the sample preparation at 4 ° C. Usually urea is added as a chaotropic agent, and nonionic detergents and protease- inhibitory substances in order to avoid interactions of the proteins and changes.

First dimension (IEF )

During the IEF ( first dimension ) of the protein extract is separated by one-dimensional, in a pH gradient gel in the electric field. The acidic and basic amino acid residues of proteins undergo depending on the environmental pH value different (de ) protonation and determine the charge of the protein. Thus, they are responsible for the effect of the electric field on the proteins. At the isoelectric point (pI), positive and negative charges of the protein abolish. PI corresponding to the pH at which the protein has a net charge of 0. There is no dynamic effect of the electric field takes on the now more charge-neutral protein and the protein deposited. By diffusion caused local changes in neighboring pH ranges lead to renewed electrical charge of the protein. The recovered effective electric field, however, promotes the protein immediately back to its isoelectric point. Two different IEF technologies are available.

Equilibration

In the so-called equilibration, which follows the separation according to the pI, the gel is initially reduced with the proteins ( for example, with mercaptoethanol or dithiothreitol ). This serves to eliminate disulfide bridges. To re-oxidation of the resultant HS -SH groups to disulfide ( -SS- ) to prevent groups in the next step, the HS- groups are alkylated with iodoacetamide, for example. Finally, the proteins with sodium dodecyl sulfate ( sodium dodecyl sulphate - SDS) laden. SDS is a negatively charged detergent. Pro 3 amino acids binds approximately one SDS molecule via its aliphatic end by hydrophobic interaction to the protein molecule. With the negatively charged side it releases itself from the loaded ends in the vicinity of bound SDS molecules. This leads to complete unfolding ( linearization) of protein molecules. The larger the protein molecule is, the longer the resulting loaded SDS chains. As a function of the length of the protein to bind a large number of negatively charged SDS molecules intrinsic charge can be ignored for most of the proteins in the following.

Second dimension (SDS -PAGE)

The gel strips with the separated and equilibrated according to the pH of proteins is placed in the vertical systems, the edge of a square or rectangular are also SDS- containing polyacrylamide gel. In horizonatelen systems, gel strip on the other hand placed a few millimeters away from the Gelrand on the flat SDS gel. The proteins are then separated perpendicular to said first dimension in a second electrophoresis according to their size. Upon application of the electric field (anode opposite the IEF gel strip ) the unfolded and surrounded by SDS proteins migrate with their excess of negative charges through the gel, which opposes the proteins according to their molecular size, a greater or lesser resistance. Small molecules migrate relatively undisturbed and quickly reach the IEF gel is remote Gelkante, large molecules are permanently braked from the gel in the migration and hardly any progress. The separation in the second dimension is facing away with the arrival of small proteins on, the IEF gel, stopped Gelrand. This can be seen by a follower dye, such as bromophenol blue. This can be added before equilibration. So that the protein pattern remains present after the separation, it needs to be fixed in a subsequent step. To various alcohols (methanol or ethanol), and acetic acid can be used. This denature the proteins separated and removed the surfactant, whereby the proteins are insoluble. Characterized diffusion is prevented and the 2D pattern is thus stable time.

SDS gel during electrophoresis

Ready- SDS gel

Mark and detect the proteins

Marker in living cells (in vivo)

Parameters such as the production of proteins in a given period ( protein synthesis ), or the phosphorylation of proteins per unit time are preferably determined by the incorporation of ( radioactive ) isotopes. For this purpose the bacteria / cell culture administered to a nutrient substrate with exceptional isotope, which is then incorporated into the proteins ( 35S ) or in the phosphate groups of phosphorylated ( 32/33P ) proteins. If the time of installation chosen to be relatively short, as a result, more of a snapshot of cellular events can be detected, with a long period of time rather the cumulative picture of many individual events. The protein extract of the labeled cells is separated and determined the proportion of labeled proteins via autoradiography or mass spectrometric method in the 2D pattern. Recently, a metabolic labeling also occur with chemically modified amino acids, which have been equipped for the subsequent labeling with fluorescent dyes with a biologically inert linker ( eg Click- iT labels). To determine the amount of protein accumulated following declared method can be used in addition to a permanent or metabolic isotope labeling on a.

Marker before the gel electrophoretic separation

Particularly for multiple samples to be separated on a 2D gel, covalently binding fluorescent dyes are used. These maximum of three different protein extracts are marked with one dye, mixed together and separated on the same gel. Since the dyes can be detected separately from each other, the generation and the differential comparison of three sample-specific protein pattern is possible ( Difference Gel Electrophoresis, DIGE ). The problem is the mass manipulation of the proteins by the bound dye. In addition, the dyes are relatively unstable and expensive.

Be used:

Marking or staining after separation

The classical protein stains take place after the electrophoretic separation. A lot of determination can be made in the non-linear dyeing process only approximately by treating the sample with proteins of known concentrations (eg, a Komigrationsstandard ). Depending on the desired specificity and sensitivity are used:

Absorbing dyes

Fluorescent dyes

By two-dimensional gel electrophoresis can be used in bacterial extracts after staining of the proteins often well over a thousand different protein species detected. In mouse embryos Klose could represent approximately 10,000 spots.

Analyze and interpret 2D gels

Digitize 2D gels

When you are digitizing 2D gels, the 2D patterns are broken down into pixels with different gray values. Resolution determines the accuracy in the x- and y-direction, the color depth the amount of gray levels, which is the mapping of the amount of protein per pixel are available.

Imaging devices

  • For all visible absorption dyes higher quality white light scanners with high Probendurchdringungsvermögen used (transmission scans with an OD to 4.0). However, without an appropriate calibration of the protein concentrations, dye signals and gray values ​​quantitative statements are later limited. Also available for digitizing the gels with visible dyes have become camera-based systems. Color filters can u.U. help improve the utilization of the dynamic range.
  • For the detection of fluorescent dyes bright laser scanners are used primarily where the wavelength of the excitation light and the optical filter for the emission light of the fluorescent dyes can be flexibly adjusted. Again, the established systems are now complemented by camera-based devices.
  • For the detection of radioactive isotopes, which have been incorporated into the proteins, so-called phosphor imager may be used. Here a so-called imaging plate is subjected to 2D gel dried for several hours. The released radioactive radiation is stored in the imaging plate and read by phosphorimager. The thus liberated from the imaging plate stored energy is linear in grayscale digital image translated (see autoradiogram above).
  • Systems for the detection of intrinsic fluorescence of proteins under UV light by excitation of aromatic amino acids such as tyrosine, phenylalanine and tryptophan could not yet prevail, but promise an interesting alternative, since no expensive fluorescent dyes have to be used. This would lead to massive time savings and in addition to the reduction of diffusion and other effects that are caused by dyeing and washing steps.

For more information on scanning of gels you can download a corresponding manual ( in English).

Single Channel techniques

In the single-channel techniques, a series of gels is added to the rule that was completely colored in the same way.

Multiplexing

The gel - multiplexing a plurality of images are generated independently from the same gel. This may be possible in different circumstances:

  • Various dyes or labels are used to represent different characteristics of the separated proteins. For example, the amount of protein can be detected by the Flamingo dye and protein phosphorylation by the ProQ Diamond dye in the same gel. Both fluorescent dyes are separately detected by the scanner and stored in two grayscale images. These can be put together with an appropriate imaging software to create a false color image.
  • Visualization of protein amount (green) and protein synthesis (red) ( Software: Delta2D ) A further possibility is the combination of an autoradiogram ( this represents the protein synthesis is determined by a pulse-labeling is radioactive ) and a protein level image ( detection of the proteins with silver staining ). In the illustrated false-color image, the accumulated proteins are visible in red the newly synthesized in green.
  • Combinations of Emerald ProQ images ( staining of proteins with sugar molecules as side chains ), Diamond ProQ images ( color of the phosphate groups on the protein) and SyproRuby ( staining of the protein amount ) in the same three color channels can simultaneously detect phosphorylation and glycosylation of proteins.

Evaluate 2D protein patterns

Classically, 2D gels are evaluated visually on light or fluorescent dyes in the UV transilluminator. Due to the complexity of protein patterns, a software-based analysis leads to reliable results. For an overview of the current status of the procedure, see

Prepare gel images

In order to analyze gel images quantitatively, they should be freed from the 2D gel typical inhomogeneous background and adjusted to artificial signals. The picture shows an example of the decomposition of a gel image into a background component, a component with artificial signals and used for further quantitative analysis spot component.

Positionally correct gel images

A long time unsolved problem was the difficult positional reproducibility of the protein patterns in 2D gel represents a possible solution was to avoid independently prepared gels.

  • The DIGE with the simultaneous separation of up to three samples per gel is a viable option, but is confronted with more than three samples with the Reproduzierbarkeitsproblem because then turn multiple gels to be compared. Since there is no satisfactory experimental solution to avoid running differences until today, had to be found at the level of software analysis a solution.
  • When you spot a pattern-matching software in the raw 2D gel image in the first step checks for protein spots using. With the help of computer-generated spot information is an attempt to find an expression profile associated partners ( spot matching ). However, weaknesses in the spot detection lead to spot allocation errors in these processes.
  • The so-called image warping ( machine vision - or - equalization ) was introduced in 2000 in the 2D gel analysis. Automatic method of image warping to use all of the available in the 2D gel images pixel information for the calculation of an image transformation that results in the best possible positional coincidence of the gel images to be compared. By positional correction an expensive spot matching is no longer necessary, since each corresponding spots already the lie at the same positions to be compared gels. Positional transformed gels measured in monetary ideal gels that do not have system-related distortions more.

Referenzgele and proteome

For a science-based interpretation of the 2D gels, the identity of the protein spots behind the proteins is determined. This can be done by different technologies, such as Edman degradation or mass spectrometry methods such as MALDI-TOF mass spectrometry. While in the 1990s, the protein spots were manually excised from the gels, today robots have found their way into the laboratories used to dig out the spots and for pipetting. Using the robotic several hundred proteins can be identified almost overnight.

Due to the introduction of computer-assisted positional correction of the protein patterns it was possible to transfer protein spot identifications easily from one gel to another without having to re- identify the spots. A 2-D gel showing the protein extract from a cell culture is from only a subset of all possible cellular proteins. Only Gelserien from cell cultures, which were grown under different growth conditions, the total number of all possible proteins can show. Reason, the differential gene expression, which only allows the production of important proteins and the synthesis currently just prevent unnecessary proteins. To construct a comprehensive Proteomekarten that contain a large proportion of all possible proteins, gel frames are positionally aligned and grouped on image fusion algorithms to yield one composite image. The composite image is combined with the data from the identification and protein can then be used as a reference for the interpretation of additional experiments.

Detect and quantify protein spots

For a ( semi) quantitative analysis of the 2D gels, the total absorption ( absorption dyes), the total radio signal ( radio-labeled proteins, the autoradiogram ) and the total fluorescence signal ( fluorescence dyes) is detected a protein spots over all image points. In the first spot detection step, the coordinates of the protein spots and in the second the corresponding spot shapes are determined. The determination of spot outlines can be done close to the pixel information, or via mathematical models. Interference information, such as Background, artifacts and image noise ( see Fig preparation) are excluded prior to quantitation. The gray values ​​of the pixels are corrected if necessary with devices and dye- dependent calibration curves and then summed inside the spot outlines found to Rohquantität. The Rohquantitäten are standardized and assigned to the corresponding protein spots. Since, as previously mentioned not spot detection from gel to gel provides absolutely reproducible results, it can come in mapping proteins to their expression profiles to errors, which sometimes can not be resolved with the established methods. Therefore, a new method for spot matching was introduced in 2003. This is based on the definition of a Spotconsensus from all the analyzed gels of an experiment on the basis of a composite image. Because the composite image contains all the information of the spot, the total experiment, the Spotconsensus can be applied at least to all those gels for quantitation spot, from which the composite image is created. Spot mapping errors can be excluded when using this method.

Visualize data

Through the use of Proteomekarten and kompositbildbasierter spot detection, completely new possibilities of visualization of protein spots that were conspicuous in the analyzed experiment result. The picture, for example, shows a summary of four different protein synthesis patterns. The colors indicate the environmental conditions under which that protein is increased at least twofold in its synthesis. With the aid of color-coding, it is possible for the first time, in addition to the positional data of the protein spots to visualize now regulation data. Biomarkers are thus quickly and reliably identified.

Modifications of the classical 2D gels

Chemistry of the release system

While the classic 2D gels proteins in the first dimension according to their isoelectric point and in the second dimension separated according to their size, other separation methods are combined in two dimensions in practice for quite proteins. Examples are:

  • The combination of two different detergents in the two dimensions.
  • "Blue native" Electrophoresis in the first and SDS gel electrophoresis in the second dimension,
  • Separation under oxic ( first dimension ) and reducing ( second dimension ) conditions

Geometry of the separation system

  • The separation of the proteins according to the isoelectric point is usually horizontal or in the gel strip and vertically long cylindrical gels, which were prepared in tubes.
  • The second separation is carried out in sandwich of two glass plates with an intermediate gel. These glass plates gel sandwich vertically suspended in batches of 1, 2, 5, 6, 10 or 12 gels in tanks containing the running buffer during the separation and are cooled by a cryostat.
  • Commercially available precast gels are often used in horizontal systems on cooling plates for protein separation. Depending on the dimensioning of 1 to 4 or more gels may be processed in parallel, too.
  • Another new example of the use of radially symmetrical setups that separate the IEF strips in the 2nd dimension from the inside out in a circular gel slices. Depending on the length of the IEF gels, up to six batches are separated on a gel slice. It is intended to stack several of these discs in a plant. Since here the electric field is from the inside to the outside, the field lines away from each other, the further the proteins migrate to the outside. This prevents that move quickly especially small proteins and leave the gel again, even before the big slow proteins were sufficiently separated.
  • To drive the parallel separation of samples on the top, geometric three-dimensional gels were developed ( www.3d- gel.com ), which can simultaneously separate 36 IEF gels on a gel block. The detection of proteins occurs, as with traditional DNA sequencers in a illuminated by a laser Gelebene. During the passage of the labeled prior to separation of proteins with fluorescent dyes emission light is detected. The measured signals are stored. The gel images are reconstructed from the stored image stack by software and conventionally evaluated.

Advantages of 2D gels

Problems of the 2D gel electrophoresis

As any other technique in the protein biochemistry also carries the 2D -gel electrophoresis has some problems:

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