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Tricine SDS-PAGE gel under non-reducing - please tell me a protocol (May/02/2006 )

Hi all,
Now I want to run my refolded protein (three disulfide bonds) by tricine SDS-PAGE gel under non-reducing conditions and then extract the band of the protein from gel for analysis by RP-HPLC. So what should I do?
Please give me the protocol of non-reducing tricine SDS-PAGE and the protocol of polyacrylamide gel extraction
Thanks biggrin.gif


Do you mean non-denaturing conditions, or non-reducing conditions?

It'd be tough to do PAGE under non-denaturing conditions if you include SDS in the gel. Non-reducing conditions I guess would mean leaving out BME or DTT from your sample buffer...

Invitrogen sells a native (non-denaturing) PAGE sytem (info here) based on the Blue-Native PAGE system (BN-PAGE) developed by developed by Schagger and von Jagow.

See Schagger, H. and von Jagow, G. (1991) Anal. Biochem. 199: 223-231 [PubMed], Schagger, H. (2001) Meth. Cell Biol. 65: 231-244 [PubMed], or Schagger, H., Cramer, W.A. and von Jagow, G. (1994) Anal. Biochem. 217: 220-230 [PubMed].