reducing and nonreducing buffer, SDS-PAGE - (Jan/19/2006 )

Hi,

can anybody tell me if we run reducing sample and non-reducing sample on SDS-PAGE, which runs fast and why.

thanks in advance

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what do you mean by which runs fast?

reducing buffer will disrupt the disulfide bond of your proteins.

instead of an homodimer of 100 kDa, you will see the monomer of 50 kDa for example.
(the reduced runs faster?)

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Generally a reduced sample is going to run faster than a nonreduced one, for two reasons.

1) Inter-chain disulfide bonds. Obviously, if you've got a disulfide linked dimer, that's going to be twice as big as the monomer species (or 3x for a trimer, etc). This one is fairly obvious.

2) The formation of nonlinear species can effect mobility - ie, if you have an intrachain disulfide bond in a monomer, it may run differently than the reduced monomer. Similarly, a disulfide-linked dimer may not run at exactly double the size of the monomer.

(In my personal experience, the closer to the middle of a protein sequence the crosslink occurs, the slower it migrates, meaning an "X" shaped molecule will appear larger than a straight line shaped molecule - I only have data for one protein species with a bunch of cys mutations. The literature pretty much stops at "nonlinear species look different from linear species").

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Thanks for clearing my doubt

suguna

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