Appendix F.  Gel Electrophoresis


Electrophoresis is the method of separating charged molecules in an electric field. Electrophoretic separations are most often performed in a semisolid matrix, such as a gel, rather than in free solution because gels enhance the separation of macromolecules, like proteins and nucleic acids. Gels enhance separation in two ways. First, gels reduce convective mixing of samples. Second, the natural pores found in gels act as molecular sieves, which act to separate molecules based on their molecular size. Thus, gel electrophoresis is the method of choice when separating macromolecules based on their molecular size.

The effectiveness of the separation of macromolecules in gel electrophoresis depends on the size of the pores in the gel. Pores that are too large will permit macromolecules of all sizes to migrate together at a high rate. Pore sizes that are two small will impede the migration of all molecules. In either case, separation of the molecules is reduced. Pore sizes that are just right will permit a migration rate that is inversely proportional to molecular weight, permitting effective separation of the molecules in the gel.

Polyacrylamide Gel Electrophoresis (PAGE)

Polyacrylamide gels are the gel of choice for separating proteins. Polyacrylamide gels are easily made by mixing acrylamide with bisacrylamide and two polymerization reagents (ammonium persulfate and TEMED). The spaces found among the interlinking acrylamide and bisacrylamide molecules are the pores that permit protein migration through the gel. Polyacrylamide gels are inert, easy to make, and their pore size can be controlled by simply adjusting the concentration of acrylamide & bisacrylamide used in making the gel (usually ranging from 4% - 15%). An electron micrograph of a polyacrylamide gel, clearly showing the gel pores, is shown below.

Sample Preparation with SDS. In polyacrylamide gel electrophoresis (PAGE), the protein sample must be prepared properly in order to separate proteins based on their molecular weight. When preparing the sample properly, three conditions must be met.

First, the proteins must be completely denatured. By denaturing the proteins, the differences in the 3-dimensional shapes of the proteins will be eliminated and therefore protein shape will not affect the migration of proteins through the gel. Proteins are easily denatured by first solubilizing the proteins in a special buffer called electrophoresis sample buffer, then immersing the treated sample in boiling water. The sample buffer contains mercaptoethanol and sodium dodecyl sulfate (SDS). Both of these ingredients aid in the denaturation of the protein; mercaptoethanol breaks all disulfide bonds in the protein, and SDS, a detergent, interfers with all hydrophobic bonds. Together, with approximately five minutes of boiling, the proteins in the sample are completely denatured.

Sample Preparation

Second, all proteins must be solubilized in an aqueous medium. This is not an issue for hydrophilic proteins, but is a consideration when dealing with membrane-bound proteins, which can be very hydrophobic. This condition can be met with SDS as well.  As a detegent, SDS breaks the hydrophobic bonds that stabilize integral membrane proteins in the membrane, thereby removing the proteins from membranes and solubilizing them in water.

Third, because there are natural differences in the net charge of different proteins due to their different amino acid compositions, differences in net charge among the different proteins must be eliminated.  Again, this is met by SDS. The SDS molecules themselves have a negative charge (SDS is an anionic detergent), and it binds stoichiometrically to the denatured proteins at a ratio of approximately one SDS molecule per two amino acids. This stoichiometric binding masks the natural charge of the proteins and gives all proteins in the mixture the same amount of negative charge per unit mass, thus removing all differences in net charge. Thus, the samples that are loaded into the wells of the gel contain a mixture of proteins that differ only in the molecular weights, and as they migrate through the gel, they will separate according to their molecular weight.

So you can see that sodium dodecyl sulfate (SDS) is a critical ingredient when using polyacrylamide gel electrophoresis (PAGE) to separate proteins based on their molecular size.

The Protein Standards

The precise mathematical relationship between relative mobility and protein molecular weight is different for each gel. So, the first step in determining the molecular weights from your gel is to determine the precise mathematical relationship between relative mobility and molecular weight on your gel. This is the purpose of the standards. The standards are known proteins of known molecular weights. Since we know their molecular weights and since we can determine their relative mobilities from the gel, we use these standards to determine the precise mathematical relationship between relative mobility and molecular weight for your gel. Therefore, a set of standards must be run on every gel produced, and one must use the standards run on one’s own gel. The standards run on someone else’s gel are not valid for your gel. 

Load Samples

Load your Sample onto the Gel. Load a prescribed volume of your sample into the assigned wells at the top of your the gel.

3. Run the Gel. Once all samples have been loaded, place the lid on the electrophoresis chamber. Attach the leads to the power supply. When assembled properly, the anode will be at the top of the gel, the cathode will be at the bottom. Apply power. The recommended power for these gels is 200 volts.

Your team needs to monitor the migration of your samples through the gel. When the solvent front migrates to within 0.5 cm of the bottom of the gel, the run is over. Turn off the power supply. The usual run time is approximately 35 - 40 minutes.

4. Stain the Gel. Remove the gel from the electrophoresis apparatus. Place the gel in a staining tray and proceed to stain the gel according to the following protocol:

CellBiologyOLM is authored by Stephen Gallik, Ph. D.| Copyright © 2011 by Stephen Gallik, Ph. D. | Licensed under a Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported License