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Three proteins A, B and C of equal molecular weight are being investigated in a study. They contain six, four and four cysteine residues respectively. Only Proteins A and B were treated with β-mercaptoethanol (which reduces disulphide bond) and heated in boiling water bath for a few minutes. Which of the following is expected in the SDS PAGE gel run?

a. Protein C will move fastest.

b. Protein B will move fastest.

c. Proteins A and B will move at the same speed but faster than C.

d. Proteins B and C will move at the same speed.

Before knowing the answer the I thought the answer would be b. Because as Protein A has the most no. of amino acids so it will be behind Protein B and C for sure. And for protein B and C, as Protein B has been in a reducing environment it will not form disulfide bridges but Protein C will, so it will be billore and hence will be behind Protein B. So B will be fastest?

But the answer key says C, I am not really sure how?

So the answer is C . Before knowing the answer I thought the answer would be b. Because as Protein A has the most no. of amino acids so it will be behind Protein B and C for sure. And for protein b and c as Protein B has been in a reducing environment it will not form disulfide bridges but Protein C will so it will be billore and hence will be behind Protein B. So B will be fastest? But the answer key says C

The problem statement says all the proteins have the same molecular weight but does not say how many amino acids they have. It does say how many cysteine amino acids each has, but not how many other kinds they have. Cysteine forms disulfide bonds, holding bends in the protein structure.

BiteSizeBio says:

SDS is a detergent that is present in the SDS-PAGE sample buffer where, along with a bit of boiling, and a reducing agent (normally DTT or B-ME to break down protein-protein disulphide bonds), it disrupts the tertiary structure of proteins. This brings the folded proteins down to linear molecules. SDS also coats the protein with a uniform negative charge, which masks the intrinsic charges on the R-groups.

So the point to SDS-PAGE is to have proteins migrate in the gel according to molecular weight without regard to the natural charges carried by the various component amino acids. The three proteins should thus migrate at the same rates in SDS-PAGE (not A being slower than B and C, as OP thought).

However, protein C is not treated with β-mercaptoethanol (B-ME) which is an important part of SDS-PAGE, so it retains the disulfide cross-links and thus is not a linear string of amino acids. Hence C will migrate more slowly than A and B, and the expected result is as stated in choice (c.).