Dear Daniel,

few considerations:

1) do you understand how heat treatment affects the multimeric state? Could it be that the dimer forms when the protein is partially unfolded? This brings me to the next point:

2) does the pH of the SEC buffer match the one of the PAGE running buffer, and how those pHs differ from protein pI?

3) Also, can you modulate the dimerization by other factors (i.e. reducing agents, glycerol, arg/glu, etc)?

4) It might also be that as the protein migrate in the gel, its local concentration is higher than in the column, which stimulates the dimerization.

5) What is the starting temperature in the PAGE running buffer?

Overall, it would be nice to know the nature of the dimerization, whether its hydrophobic (which would speak for unfolding), or electrostatic (the pH is responsible), or is induced by the disulfide bond formation, or simply by the concentration effect.

Hi Anton, excellent questions/comments.

1) heat appears to promote dimerizarion.

2) SEC is run at RT and uses pbs 7.4. Native page uses running buffer at 8

3) not sure if thise aforementioned reagents affects dimerization.

4) I think that may be correct. I will try loading a gradient concentration to see if this makes a difference.

5) although I run the gel at 4 degrees, the buffer starts at RT. Perhaps next time I will chill the buffer before running.

The dimer is held together via hydrophobic intermolecular forces. There are no cysteines in the sequence.

your comments have helped me already thank you very much!

Hi Daniel, glad to be of help :)

I wonder now what would happen if SEC is done in pH 8, or even in tris-glycine buffer, same as in native PAGE.

Are you running a continuous or a discontinuous electrophoresis? In the latter case, the pH the protein is exposed to during the run will be quite different from that of the sample or gel buffers.