A major requirement for separation techniques is high resolution. The separation technique should produce fractions that comprise very simple mixtures of proteins, and ideally each fraction should contain an individual protein. This essentially rules out 1D techniques i.e. those that exploit a single chemical or physical property as the basis for separation. The other major requirement in proteomics is high throughput. The separation technique should resolve all the proteins in one experiment and should ideally be easy to automate. The final requirement is that the fractionation procedure should be compatible with downstream analysis by mass spectrometry. The two groups of techniques which dominate proteomics is 2DGE and multidimensional liquid chromatography.
Any charged molecule in solution will migrate in a applied electric field, a phenomenon known as electrophoresis. The rate of migration depends on the strength of the electric field and the charge density of the molecule, i.e. the ratio of charge to mass. Polyacrylamide gels are favored because they facilitate separation by sieving the proteins on the basis of their size.
All high resolution protein fractionation methods employ multidimensional separation processes that exploit different properties of proteins for separation in each dimension. Although PAGE separates proteins according to both charge and mass, exploiting both these principles in the same dimension still results in a low resolution separation.
First dimension is usually IEF, in which proteins are separated on the basis of their net charge irrespective of their mass. The underlying principle is that electrophoresis is carried out in a pH gradient, allowing each protein to migrate to its pI, the point at which its pI is equivalent to the surrounding pH.
IPG gels are the most commonly used since they don’t suffer from cathodic drift and poor reproducibility.
The second dimension, usually carried out by SDS-PAGE, separates the proteins according to the molecular mass irrespective of charge. SDS binds to the proteins, dwarfing protein charge density.
CE is carried out in glass tubes that are typically about 50 micrometer in diameter and up to 1 meter in length. The tubes may or may not be filled with gel, but the presence of gel facilitates sieving of the proteins or peptides and enhances size-independent separation. The thin tubes are efficient at dissipating heat, allowing the use of very strong electric fields. The separations are thus rapid, efficient and can be monitored in real time rather than at the experiment’s end point. Therefore, the major application of capillary electrophoresis has been the separation of peptides in relatively simple mixtures such as tryptic digests of purified proteins or spots excised from 2D gels.
Recently CE is coupled with HPLC and ESI MS/MS for high throughput proteomics.
Major limitations are:
- Resolution
- Sensitivity
- Representation
- Automation
There are several ways to increase the resolution of 2DGE:
- Use a larger gel
- Use zoom gels – 2nd dimension on 2 gels
- prefractionation
There are many different techniques to stain gels but there is no single ideal method. The choice often depends on your needs often lead to a combination of techniques. While several reactive dyes could be used to stain proteins prior to electrophoresis, the post-electrophoretic staining of separated proteins remains the preferred method.
Proteins which are inherently which are inherently color such as hemoglobin and myoglobin are easily observed directly in polyacrylamide gels upon exposure to light in the visible spectrum. Unfortunately, this is not the case for the vast majority of proteins whose visualization requires the use of dyes or stains. Many of the organic dyes and stains that have been adapted fro the detection of proteins in polyacrylamide gels have been derived from dyes originally utilized in the textile industry. Currently the most commonly used organic dyes include amido black, coomassie blue and silver staining.
Coomassie blue
CBB-R (reddish blue) and CBB-G (greenish blue) are the most sensitive and convenient to use. CBB requires an acid environment to enhance ionic interactions between dye and basic amino acid moieties of the protein. The proteins and CBB interact via hydrogen bonds and van der waals forces.
Duration of staining depends on gel thickness and polyacrylamide composition. Thicker the gel, longer the staining time. Gels can be stained by either passive diffusion or electrophoretic destaining.
CBB is an aromatic compound. It is not suitable with SDS if you wish to store the gel for a long period of time.
Amido Black
Amido black is not as sensitive as CBB but it enjoys selected applications because of its rapid staining and destaining properties.
Silver Staining
For most applications, visualization of proteins with CBB is sufficiently sensitive. However, if one is interested in determining the absolute purity of a protein or wishes to determine trace amounts of proteins then more sensitive protein-visualization techniques must be utilized. [5]
Although silver staining is the most sensitive of all non-radioactive protein-visualization methods currently available, it does have a number of drawbacks. Probably on of the most serious problems, and one that deserves mention, is the fact that certain proteins stain either very poorly or not at all with silver, appearing as negatively stained spots against a darker background. The shortcoming is further emphasized by the lack of staining of certain calcium-binding proteins, such as calmodulin. [5]
Fluorescent protein labeling
Fluorescent labeling methods are extremely sensitive but not commonly used largely due to the difficulty of use, need for additional equipment, and cost. Although proteins can be label after electrophoresis, they are usually labeled before electrophoresis. Fluorescence molecules bind covalently to proteins. Commonly used fluorophores are dansyl and ans.
Reverse staining
The idea behind reverse staining is to stain the gel and not the proteins. Staining gels are rapid, display intermediate sensitivity and do not require prior fixation of proteins within the gel matrix.
Labeling proteins with radioactive isotopes
Radiolabeling methods
Labeling proteins with radioactive isotopes before or after electrophoresis is the most sensitive labeling method. The commonly used isotopes are C14, S35, P32, H3, and I125. Cell or tissues are exposed to radioactive isotopes which they metabolize. Alternately, Proteins are labeled post-synthetically by any of a variety of chemical methods including oxidative iodination.
Radiolabeled protein bands or spots can be detected by liquid scintillation counting or autoradiography.
Immunoblotting
Using antibodies to spot desirable proteins.
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[3] Sanchez, ARBF 1998, Sample preparation and solubilization: crucial steps preceding the two-dimensional gel electrophoresis process
[4] R.M. Twyman, Principles of Proteomics
[5] Peter J. Wirth, Alfredo Romano, 1995, Staining methods in gel electrophoresis, including use of multiple detection methods
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