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

(Last Updated On: October 28, 2022)
APS and temed
Chemical structures of Ammonium persulfate and TEMED

What are APS and TEMED?

Before jumping into the uses of the APS and TEMED in SDS PAGE, let’s know more about the individual components. APS is the ammonium persulfate while TEMED is the N, N, N’, N’-tetramethyl ethylene diamine. In the same manner, SDS is sodium dodecyl sulfate and PAGE is the poly acryl amide gel electrophoresis. SDS and PAGE together are also called denaturing poly acryl amide gel electrophoresis

APS and TEMED are the polymerization starting components basically used in the polyacrylamide gel electrophoresis. During the PAGE, 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.

Mechanism of action of APS and TEMED in SDS PAGE

APS is a free radical generating component which upon catalyzed by the TEMED generates sulfate free radicals. It is the one that initiates the polymerization of acrylamide and bisacrylamide. TEMED is added at the last in the gel mixture. when TEMED is added to the mixture, It reacts with the ammonium persulfate and causes the 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.

What is an Electrophoresis?

It 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 an equal mass-to-charge ratio so as to allow the protein to move constantly through the gel based on its molecular weight. In SDS-PAGE, 2-mercaptoethanol is also added to cleave the disulfide linkage if any is 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. After reading this post, I hope you will be able to understand the uses of APS and TEMED in SDS PAGE.

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