Use of the APS and TEMED in SDS-PAGE

Structure of ammonium persulfate

A structure of the ammonium persulfate (APS). Image: Żbiczek via Common Wikimedia

APS and TEMED in SDS-PAGE are used to polymerize the Acrylamide gel. During electrophoresis by SDS-PAGE, Acrylamide and Bisacrylamide are used to form a gel matrix. Acrylamide forms a polymeric chain like structure in which bisacrylamide creates a cross linking.  Thus, it gives the overall gel-like matrix with varying pore size depending upon the concentration of bisacrylamide.

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APS or ammonium persulfate is the one which causes polymerization of the Acrylamide and bisacrylamide when acted upon by the TEMED (TetraMethylEthyleneDiamine) and the polymerization is based on a free radical mechanism. TEMED reacts with the APS splitting the APS into the sulfate free radical which then initiates the polymerization of the Acrylamide and Bisacrylamide.

An electrophoresis is an analytical tool used in the biochemistry laboratory to separate proteins, DNA and RNA from the sample and their characterization. There are different types of electrophoresis like capillary electrophoresis, agarose gel electrophoresis, polyacrylamide gel electrophoresis, etc.

structure of TEMED

A structure of the TEMED. Image: Edgar181 via Common Wikimedia

SDS-PAGE is a denaturing polyacrylamide gel electrophoresis, frequently used in the biochemistry laboratory to separate and characterize proteins and enzymes. 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, 2-mercaptoethanol is also used 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. In the non-denaturing condition, proteins are separated in their native structure and different proteins with their intact subunits migrate through the gel.

Not only in the SDS-PAGE, but also in the non-denaturing polyacrylamide gel electrophoresis, APS and TEMED are used. 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 ray, it can generate free radicals to initiate polymerization of the Acrylamide and bisacrylamide and the process is called as photopolymerization.

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