Oh! I see. In that case, serum albumin migrates differently under reducing and non-reducing conditions (it will migrate at higher mw when reduced), antibodies will dissociate into the heavy and light chains under reducing conditions (although the reduced species will migrate at lower mw), and so on. It is hard to say without more details.

There is another alternative: that intermolecular disulfide bonds are being re-formed as the protein mixture enters the gel. Why don't you reduce all disulfides and alkylate them with something like iodoacetamide to rule this out?

I would suggest that addition of 2-mercaptoethanol leads to an improved denaturation of the proteins in your sample (and probably to the dissociation of protein aggregates that are otherwise unable to enter your gel at all). That could explain why you see some protein bands in the higher molecular range when you reduce your sample.

Best regards

Just to clarify. You mean you have a purified protein that migrates as a single band in non-reducing SDS-PAGE, but the same preparation exhibits several higher molecular weight bands under reducing conditions?

A picture of your gel would be useful here.

Dear Mr.Martin

I have a protein isolate, which is a mixture of 4 albomine, globuline,... fractions. we didint work on the protein fractions. We worked on the SDS-PSGE pattern of this protein isolate in 2 conditions. The results showed in reduced condition the number of bands are more then what appears in non-reduced one. Actually they moved to higher molecular weight and also the derivated. 

I'll be thankful if you guide me with the articles who worked on this subject and prove it or reached to the same point.

Oh! I see. In that case, serum albumin migrates differently under reducing and non-reducing conditions (it will migrate at higher mw when reduced), antibodies will dissociate into the heavy and light chains under reducing conditions (although the reduced species will migrate at lower mw), and so on. It is hard to say without more details.

There is another alternative: that intermolecular disulfide bonds are being re-formed as the protein mixture enters the gel. Why don't you reduce all disulfides and alkylate them with something like iodoacetamide to rule this out?

Thank you dear Martin. I have no sample anymore. My sample is a plant protein isolate, actually it is obtained from a legume seed. I observed  this events. Now I need document explanation as an revise to the reviewers of my article. My finding was that I explained in my question, but it seems they want me to make it more clear... because of that I need to read and present a few other studies.