Proteomics/Protein Separations- Electrophoresis/Gel Electrophoresis

Theory
Gel electrophoresis (GE) is used to differentiate molecular entities depending on their physical characteristics. These defining characteristics include the size, shape, charge and isoelectric point. Isoelectric point is perhaps the most important characteristic; it is the point at which the overall electric force on the analyte is 0, and hence the point at which the analyte ceases migration within the gel. Gel electrophoresis is used as an analytical technique or as a preparatory technique to purify molecules before they are used for other methods like mass spectrometry is based on the principle that, when charged molecules are placed in an electric field, they migrate toward either the positive or negative pole depending on their charge. Since nucleic acids are negatively charged due to their phosphate groups they migrate toward the anode. Unlike nucleic acids, since proteins can have either a net positive or a net negative charge they can migrate to either of the poles depending on the charge.

The shape and charge of proteins have a dramatic affect on protein migration behavior during gel electrophoresis, especially if the protein is not denatured prior to electrophoresis. Polyacrylamide gel electrophoresis (PAGE) is commonly used separating proteins. PAGE can be used to purify proteins prior to other proteomics techniques or to analyze information on the mass, the charge on proteins, and/or presence of a protein. Due to these complex structures, proteins are usually denatured, or broken down to simple primary structures in the presence of a detergent such as sodium dodecyl sulfate (SDS), which imparts a negative charge on proteins, and thus allows for proper migration. The quantity of SDS bound and the size of the protein are relative to each other, thus this method separates proteins mainly based on molecular weight. Two-dimensional PAGE (2-D PAGE) differentiates proteins in the first dimension by isoelectric point and in the second dimension by molecular weight. Native PAGE separates proteins by mass/charge ratio without denaturing them.

Gel Properties and Preparation
Electrophoresis is usually carried out within a matrix or gel made of agarose or polyacrylamide. These gels are chemically inert, so they will interfere little with analytes. Agarose is a polysaccharide, an extraction from seaweed. The concentration of agarose used in any particular gel can vary. As a rule, the higher the concentration of agarose in the gel, the smaller the pore size will be.

For separation of proteins, polyacrylamide (a cross-linked polymer of acrylamide) is used. To avoid inhibition of polymerization by oxygen, the liquid polyacrylamide is poured between glass plates to make gel slabs. Low concentrations of polyacrylamide or less cross linking between its component acrylamide molecules results in gels with large pores. Standard protein gels are typically composed of two layers, a topmost layer called the stacking gel, and a lower layer, which is called a resolving or separating gel. The stacking layer contains a low percentage of acylamide and low pH. The acrylamide concentration of the separating gel varies according to the samples to be run, and its pH is higher than the stacking gel. The difference in pH and acrylamide concentrations between the stacking and separating gels provides better resolution and sharper bands in the separating gel. Polyacrylamide gels have a high resolving power compared to agarose gels.

Acrylamide gels are frequently used to separate DNA, RNA, oligonucleotides, and other smaller molecules due to its less porous nature. Using different concentrations of aryclamide provides a thicker or thinner matrix. As with any gel, the concentration of the gel or unique characteristics of its composition may affect migration of analytes.

Separation
Gels contain wells in which to place samples, and analytes start their migration from these points. A molecular marker can be run in one or both sides of the gel for later comparison, because it will migrate through the gel with the samples once electric current is applied. The electrophoresis buffer which covers the gel provides uniform pH and provides ions to support conductivity. When an electric current is applied across the gel, the sample molecules start moving through the gel matrix at different rates, towards the cathode if positively charged, or towards the anode if negatively charged. The voltage, as well as the time allotted for a particular run, depends on the size of the gel and the type of sample being analyzed. Running the current too long may cause samples to migrate off the gel, and turning the voltage too high may damage the samples or burn the gel itself.



Visualization and Analysis
It is rare that analyte fragments can be seen by the naked eye within gels, so gels must be stained to allow for analysis. Analyte fragments resolve as solid bands within the gel. A variety of stains can be used to view bands, including ethidium bromide, silver, or Commassie blue, depending on the sample. Less common stains that may be used are fluorescent dyes, zinc, and copper. Staining times can vary from a few hours to a few days, depending on the type of gel and the type of analyte. The resolution of stains can also vary.

Visualization can be done by the naked eye once staining is complete. It is often easier, however, to use ultraviolet (UV) light to provide easier visualization. This method, however, only works if the molecules being considered fluoresce under UV light. Alternatively, an autoradiogram can be recorded of the gel if sample molecules contain radioactive atoms.

Analysis of bands consists of comparing those generated by the sample to those from a known protein, be it a control sample or a molecular marker (standard). Further analysis can be done by removing a piece of the gel containing a band of interest. In-gel digestion can remove the protein from the smaller gel piece and allow for direct manipulation of the analyte. Analysis of gels are often time-consuming, and it is difficult to get distinct bands because samples are rarely pure.