What are the uses of APS and TEMED in SDS-PAGE protocol?

(Last Updated On: July 20, 2021)
ammonium persulfate and temed
Chemical structures of Ammonium persulfate and TEMED

APS and TEMED are the polymerization starting components basically used in the polyacrylamide gel electrophoresis. During the polyacrylamide gel electrophoresis, acrylamide and bisacrylamide are added to form a gel matrix. Acrylamide forms polymers in which bisacrylamide creates a cross-linking.  Thus, it gives the overall gel-like matrix with varying pore sizes depending upon the concentration of bisacrylamide.

APS or ammonium persulfate is the one that initiates polymerization of the acrylamide and bisacrylamide. TEMED, the full name of which is (N, N, N’, N’ Tetramethylethylene-1,2-diamine is added at the last in the gel mixture. It reacts with the ammonium persulfate and causes splitting of the persulfate ions into sulfate free radicals. Thus formed sulfate free radicals now initiate the polymerization (free radical reaction) of the acrylamide and cross-linking with bisacrylamide.

Electrophoresis is an analytical tool used in the biochemistry laboratory to separate and characterize proteins and nucleic acids based on their net molecular charge to mass ratio. There are different types of electrophoresis like capillary electrophoresis, agarose gel electrophoresis, polyacrylamide gel electrophoresis, etc. Polyacrylamide gel electrophoresis can be of two types; denaturing and non-denaturing.

SDS-PAGE is a denaturing polyacrylamide gel electrophoresis, frequently used in the biochemistry laboratory to separate and characterize proteins. In this type of electrophoresis, sodium dodecyl sulfate (SDS) is used to denature the protein and provide equal mass to charge ratio so as to allow the protein to move constantly through the gel based on their molecular weight. In SDS-PAGE, 2-mercaptoethanol is also added to cleave the disulfide linkage if any present.

Electrophoresis can also be carried out in non-denaturing conditions, without using denaturing agents like SDS and 2-mercaptoethanol. This is called a non-denaturing PAGE or Native-PAGE. In the non-denaturing condition, proteins are separated in their native structure and different proteins with their intact subunits migrate through the gel.

APS and TEMED are not only used in the SDS-PAGE but also in the native PAGE. However, in some cases, instead of using APS and TEMED, riboflavin can be used as a polymerization starting material. Riboflavin is a part of vitamin B and is photosensitive. In the presence of UV rays, it can generate free radicals to initiate polymerization of the Acrylamide and bisacrylamide and the process is called photopolymerization.

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