I am using cytiva microspin G25 columns to get rid of excess unicorporated ATP from my protein sample. The protein is 28kDa. The issue that I am facing is that after following the Cytiva protocol to recover the sample from the column, I am not getting any protein out in the eluent of G25. I recover almost the same volume I load but it does not have any protein in it (as evident from the SDS-PAGE I ran).
If somebody can provide some insights into why this might be happening and possible solutions, I'd appreciate the help.
Thank you.
Your protein may have an affinity for the carbohydrate resin. Try a Bio-gel spin column instead (e.g. Bio-Rad micro Bio-Spin P-6). It is made of polyacrylamide.
What Adam said! Or - if you are changing buffer - you protein may not be soluble in the new buffer.
Yes, and buffer additives (e.g., detergent, salt, chaotrope, HOAc) can help block the non-specific interactions Adam mentioned, as long as they do not cause solubility problems, as Bo idicated.
Dear Kriti, sometimes blocking the column with another protein like BSA can reduce unspecific adsorption/binding so Sephadex resins. Also adjusting salt concentration sometimes helps.
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