Explain the purpose of the components of an acrylamide gel. Which components of

Question

Explain the purpose of the components of an acrylamide gel. Which components of an acrylamide gel affect the qualities of the final product, and which components effect the speed of polymerization? If you run Dalton VII mixed markers on an acrylamide gel and only five bands show up, what are some explanations for what happens t o the other two? What is the purpose of running both Dalton VII mixed markers and lanes with individual markers on SDS-PAGE? Given the results that you see with the Dalton markers, is it possible to use a 12% separating gel to run a set of proteins that range in molecular weight from 10,000 to 200,000? Explain. What causes a protein to migrate on SDS-PAGE in such a way that it appears to be a protein that is much bigger? For a list of websites related to the material covered in this chapter, go to web connections at the Experiments in Biochemistry site on the Brooks. Cole Publishing website you can access this page at http://www. and follow the links from the chemistry page.

Explanation / Answer

SDS-PAGE (sodium dodecyl sulphate-polyacrylamide gel electrophoresis) is commonly used in the lab for the separation of proteins based on their molecular weight. It’s one of those techniques that is commonly used.

SDS-PAGE separates proteins according to their molecular weight, based on their differential rates of migration through a sieving matrix (a gel) under the influence of an applied electrical field.

The movement of any charged species through an electric field is determined by its net charge, its molecular radius and the magnitude of the applied field. But the problem with natively folded proteins is that neither their net charge nor their molecular radius is molecular weight dependent. Instead, their net charge is determined by amino acid composition i.e. the sum of the positive and negative amino acids in the protein and molecular radius by the protein’s tertiary structure.

So in their native state, different proteins with the same molecular weight would migrate at different speeds in an electrical field depending on their charge and 3D shape.To separate proteins in an electrical field based on their molecular weight, we need to destroy the tertiary structure by reducing the protein to a linear molecule, and somehow mask the intrinsic net charge of the protein. That’s where SDS comes in.

SDS also coats the protein with a uniform negative charge, which masks the intrinsic charges on the R-groups. SDS binds fairly uniformly to the linear proteins (around 1.4g SDS/ 1g protein), meaning that the charge of the protein is now approximately proportional to its molecular weight.

In an applied electrical field, the SDS-treated proteins will now move toward the positive anode at different rates depending on their molecular weight. These different mobilities will be exaggerated the high-friction environment of an gel matrix.

MIGRATION IS MAINLY DUE TO APPLIED ELECTRIC FIELD AND PROTEINS ARE SDS TREATED HENCE WEIGHT IS APPEARS MORE.

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