Discussion
A protein mixture contains three polypeptides A, B and C, whose masses are 63, 28 and 79 kDa with pI values of 6.5, 7.0 and 8.0, respectively, were subjected to standard reducing SDS-PAGE. The order of their separation from top to bottom would be :
Answer: D
From top to bottom the order is C, A and B.
SDS coats denatured polypeptides with a uniform negative charge in proportion to their length, and the reducing agent breaks disulfide bonds. Charge differences, and so pI, therefore stop mattering: the gel separates purely by mass, with the smallest polypeptides moving fastest and furthest.
So the largest, C (79 kDa), stays nearest the top, then A (63 kDa), and the smallest, B (28 kDa), runs lowest. Orders based on the A, B, C labels or on the pI values (6.5, 7.0, 8.0) apply a criterion that SDS-PAGE ignores.
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