SDS Page question

[laballsummer]

Hey guys, quick question about SDS page and reducing conditions (these questions seem to pop up a lot on both B/B and C/P), just trying to make sure I understand these concepts.

So, let's consider a protein that is a homodimer linked via disulfide bridges. If this is run in a gel in non-reducing conditions, it will migrate as the homodimer, correct?

On the other hand, if the same homodimer protein had no disulfide bridges, running it in non-reducing conditions would have it migrate in the momonermic form, correct?

So, the phrase "reducing conditions" specifically refers to BME or DTT, which will disrupt disulfide bonds? Even though, technically, SDS is, in and of itself, a reducing agent that will disrupt quaternary structure.

Thanks!

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[aldol16]

Yes, if you run something that's connected via disulfide bridges in an SDS-PAGE without reducing it, those disulfide bonds are not going to break.

Reducing conditions refers specifically to the presence of a reducing agent that is capable of reducing disulfide bonds. DTT is a good example. SDS is not a reducing agent - it is a surfactant.

 

[victorias]

But if you run the homodimer protein had no disulfide bridges, running it in non-reducing conditions, wouldn't it still have other types of bonds and so, it would not be necessarily in the monomeric form?

 

[aldol16]

But if you run the homodimer protein had no disulfide bridges, running it in non-reducing conditions, wouldn't it still have other types of bonds and so, it would not be necessarily in the monomeric form?

SDS is a denaturing agent. You want to denature the protein before you run it on the gel, otherwise migration time/length will be determined by the secondary/tertiary/quaternary structure.