Gel filtration separates on the basis of molecular weight, but also shape. If the protein has two different conformations as a result of exposure to a high concentration of ammonium sulfate during phenyl-Sepharose chromatography, it could show two peaks.

Hi Arun,

I'm assuming that ammonium sulphate is present in the starting buffer of your phenyl sepharose step, and not in your original sample. What gel filtration column are you using? If your protein aggregates, it should come out in the void volume. Have you tested both peaks on a SDS-PAGE gel?

Best regards,

Arthur

Gel filtration separates on the basis of molecular weight, but also shape. If the protein has two different conformations as a result of exposure to a high concentration of ammonium sulfate during phenyl-Sepharose chromatography, it could show two peaks.

I think the gel filtration beads also have some charge (however negligible). So, It is always advise to use any salt (generally NaCl) is the mobile phase. The salt will going to bind/mask this charge and thus, charge doesn't interfere in separation of protein molecules having very near molecular weight.

I used 0.5M Nacl in elution buffer for gel filtration.

I have tested all the fractions in SDS-PAGE, the profile is same.

Amm.sulphate was only in starting buffer for binding of sample to pheny sepharose column. sample eluted in presence of NaCl and absence of amm.sulphate.

First, have you checked whether the content of the two peaks (100 kDan and 120 kDa) are the same protein (e.g. on SDS PAGE and WB, not size but identity)? Is this 100 kDa or 120 kDa the size of your monomer or polymer (di-, tri-, tetra-, etc)? If the they were separated on an HIC column, they should be either different proteins or different polymeric state (which each will have their own hydration surface area), or the same protein but fold differently (particularly if you do refolding, one of them may be the molten globule). What would happen if you premix the two proteins separated in HIC column and load the mixture onto the GF column, I predict that you would see only one peak as well.

About the GF, I am a bit more skeptical. Proteins of similar molecular mass and shape (as Adam mentioned) may co-migrate upon elution in a GF column. You may notice that the Gel Filtration marker is unevenly spread, e.g. from BioRad consists of proteins of 1.5 kDa, 17 kDa, 44 kDa, 158 kDa, and 650 kDa. Thus, at a size above 100 kDa and below 10 kDa, I am not too keen on its precision. You can try to calculate back from your equation, at that molecular weight what the retention time would be, then compare it with the retention time you measured. It is significant if the difference in the retention time is more than 10-15%.