SDS-PAGE to detect monomer vs dimer? - How to best do this? (Dec/06/2005 )

I am trying to determine if I have both a monomer and a dimer in my protein mixture, and I would like to get up an up-front answer by SDS-PAGE. I am used to the technique of adding SDS+DTT, then boiling the protein, and then running it in a Tris/Glycine/SDS Gel. The combination of these steps leads to the detection of a monomer only. I am wondering what steps I should eliminate in order to detect the presence of a dimer, but still get a reasonable estimate of Mw? Should I just take out the DTT? Should I still boil the protein, do I still need to add SDS?

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you should have a non denaturing PAGE. So no sds and no Dtt...

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hello,

if you eliminate DTT in your sample loading buffer, you will keep disulfur bonds.
if you also eliminate boiling, you may keep other protein interactions.
I would say you can keep SDS, you will have the relative molecular weight of your dimer.

good luck

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If you're working with a recombinant protein, the best and the simpliest way is to cross-link the sample with any thiol-cleavable reagent (like DTSSP from Pierce), then incubate the sample in SDS sample buffer without DTT at room temperature, then run the gel. If there are dimers (or larger) you'll see them, and they will brake down to monomers if you treat the sample with DTT at 95C (it'll cleave the DTSSP).

If you're working with a lyzate then you can do native gel, yes, but there is no way you can estimate MW of the protein using a native gel. In the best scenario, i.e. if the dimer has reasonably different overall charge than the monomer (which is unlikely) you'll see two bands, but you'll never be able to state: look I've got a dimer! You'll say: look my protein pool is heterogenuous as far as the charge of the protein is concerned. So the best way to check for dimers in lysates is to do gel filtration, and then PAGE the fractions.

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