Gels with an acrylamide concentration gradient (gradient gels) are also used. In general, an acrylamide concentration between 6 and 15% is used. Using a higher acrylamide concentration produces a gel with a smaller mesh size suitable for the separation of small proteins. Use an appropriate gel concentration for your target protein. Proteins migrate at different rate depending on the concentration of the separating gel. Allow acrylamide to polymerize for 20-30 minutes to form a gel. Overlay with water to prevent contact with air (oxygen), which inhibits polymerization. Pour acrylamide solution for a separating gel. Thoroughly clean the glass plates with ethanol, and assemble the gel casting mold. Use an appropriate comb depending on the sample size.Įxample: Use an 8-lane comb for 7 samples and molecular weight markers. Gather combs, glass plates, spacer (silicone tubing), and binder clips.Ī comb is used to make wells (lanes) to load samples. Preparation of polyacrylamide gel ※An example performed at MBL Polyacrylamide gel electrophoresis of SDS-treated proteins allows researchers to separate proteins based on their length in an easy, inexpensive, and relatively accurate manner. The strength of the gel allows easy handling. Polymerized acrylamide (polyacrylamide) forms a mesh-like matrix suitable for the separation of proteins of typical size. In the presence of SDS and a reducing agent that cleaves disulfide bonds critical for proper folding, proteins unfold into linear chains with negative charge proportional to the polypeptide chain length. SDS is a detergent with a strong protein-denaturing effect and binds to the protein backbone at a constant molar ratio. In SDS-PAGE, the use of sodium dodecyl sulfate (SDS, also known as sodium lauryl sulfate) and polyacrylamide gel largely eliminates the influence of the structure and charge, and proteins are separated solely based on polypeptide chain length. Other influences on the rate of migration through the gel matrix include the structure and charge of the proteins. When proteins are separated by electrophoresis through a gel matrix, smaller proteins migrate faster due to less resistance from the gel matrix.