Protocols, optimization tips,
and more for Western Blot.
SDS-PAGE is the most widely used method for separating proteins by their relative molecular weights. By denaturing proteins in the presence of SDS, an ionic detergent, proteins can be linearized and imbued with a negative charge. This allows for an electric field to press them through the polyacrylamide gel matrix. Larger proteins migrate slower than small proteins, resulting in a size-dependent separation of proteins. In order to obtain picture-perfect separation, the gel concentration and running conditions must be tuned to the protein being investigated.
The concentration of the gel is largely determined by two factors: the concentration of acrylamide used, and the ratio of acrylamide to bisacrylamide. Higher concentrations of acrylamide form denser gels with smaller pore sizes, which is ideal for separating low molecular weight proteins with high resolution. Bisacrylamide is responsible for forming crosslinks between acrylamide molecules; altering the ratio of acrylamide to bisacrylamide can be used to finely adjust the pore size of the gel.
Other factors to consider include gels with varying pore sizes. A common example is the use of a stacking gel and a resolving gel; the stacking gel is a low concentration gel used to form the wells of the much higher concentration resolving gel. This allows more proteins to concentrate at the dye front before separating, allowing for the comparison of protein samples of different concentrations on the same gel.
Optimize your gel concentration based on the size of the protein you are trying to resolve. Use this handy table to aid your estimates:
|Protein MW Range||Gel Concentration|
Keywords: Western blotting optimization, SDS-PAGE, gel percentage, gel concentration, pore sizeClick for more optimization tips