Dimer or monomer - (Nov/25/2009 )

Hi, i tried the following way to determine trimeric nature of my protein and it worked --

1.Go for normal SDS-PAGE but without adding DTT or beta-mercaptoethanol to sample loading buffer.

SDS is only a denaturant agent and breaks only non-disulfide bonds responsible for tertiary andquatnary structure of protein but DTT or beta-mercaptoethanol breaks disulfide bonds which cuases oligomeric protein to appear monomeric in Reducing (i.e. withDTT or beta-mercaptoethanol) SDS PAGE.
Actually my protein's trimeric structure was due to disulfide bonds between cysteine residues.

I found all three forms - monomer,dimer and trimer with respective weights

2.I also tried Native PAGE (i.e. without DTT or beta-mercaptoethanol and without SDS in gel or any of the buffers) but as Native PAGE separates acco to combination of charge and mass (unlike uniform -ve charge provided by SDS in SDS PAGE and separation acco to size only) so molecular weight of the protein could not be determined even though marker was also run which was not necessary because marker contained DTT and did not correlate with size of protein bands.
u can only see 2 or 3 bands acco to your protein but u cant determine their size in Native PAGE.


I think u should try first method.

Amit Kumar on Wed Mar 28 21:17:08 2012 said:

You should possibly check the post dates on the thread - last replied to in 2009, so i guess the answer has already been found...