Polyacrylamide gels are formed by the reaction of acrylamide and bis-acrylamide (N,N’-methylenebisacrylamide) that results in highly cross-linked gel matrix. The gel acts as a sieve through which the proteins move in response to the electric field. Proteins contain an overall positive or negative charge; this enables the movement of a protein molecule towards the isoelectric point at which the molecule has no net charge. By denaturing the proteins and giving them a uniform negative charge, it is possible to separate them based on the size as they migrate towards the positive electrode.
Figure 1.SDS-PAGE of protein samples and color burst protein marker(C1992)
NOTE: Acrylamide and bis-acrylamide are neurotoxic in nature. All the steps should be performed wearing powder-free gloves.
*TEMED must be the last ingredient added
Coomassie Blue staining: Staining of protein gels with Coomassie Brilliant Blue R-250 is a common procedure to visualize proteins resolved by SDS-PAGE. It is highly sensitive and is suitable for long-term storage of the gels.
We offer a highly sensitive protein detection silver stain kit suitable for SDS-PAGE gels. The following reagents are additionally needed:
For double staining, stain the gel using CBB R-250 followed by silver stain using the procedures above.
Fluorescent Stains: We offer fluorescent SYPRO and Lucy stains for protein electrophoresis. The gels can be either immersed in the fluorescent stain in dark or the gel can be mixed with cathode buffer during the electrophoresis.
The staining protocols will depend on the stain being used and may be found here:
Reversible gel stains allows the user to proceed to western blotting of proteins after SDS-PAGE.
R-PROB staining: R-PROB is a unique stain that detects proteins on PAGE gels and western blots.
Copper staining: To confirm the migration and separation of proteins, the gel may be stained with a reversible stain such as CuCl2.
Figure 2.Blotting and Vertical Electrophoresis System