Protein PAGE can be performed in three ways: without SDS (\"native PAGE\"), and

Question

Protein PAGE can be performed in three ways: without SDS ("native PAGE"), and SDS-PAGE with or without a reducing agent such as dithiothreitol (DTT) or 2-mercaptoethanol (2ME). What are the effects of each of these reagents? How do they affect separation? SDS-PAGE is usually used for analysis of protein samples, not for the actual purification. Why not? You are trying to purify Your Very Favourite Protein (YVFP), which is a dimer of identical 40-kD subunits and has a pI of 6.0. After an ammonium sulphate precipitation, you run a 2-D gel on the product and determine that the major contaminants are as follows: (Contam 1) 35 kD, pI 7.3: (Contam 2) 50 kD, pI 6.0: (Contam 3) 20 kD, pI 5.9. Which of the following would be the most logical next step in the purification process? Why? a. Gel Filtration b. Anion Exchange c. Cation Exchange d. Another ammonium sulphate precipitation

Explanation / Answer

Ans 1:- Native protein page is mainly affected by charge and size as the molecules in a native protein mainly depends on it. In native proteins when the negative charged ions reach the gradient gel,their mobility is reduced and the proteins are separated.

In SDS-page it is mainly affected by molecular mass . Hence the protein are denatured and their conformatios are disrupted due to negatively charged ion after separatio.

Ans 2 ) SDS-page is usually used for analysis of protein samples and not for actual purification because of the following reasons:-

a) They do not produce other modified form of conformation for protein.

b) Their charge remains equal for all ions present in the sample thereby making it difficult for actual purification of sample.

Ans3) The next logical step in the purification process will be gel filtration because it separates the molecules according to different size as they pass through a gel filtration chamber. Here the four contaminants have different size so they can easily be separated from the samples through gel filtration.

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