Biotin-Streptavidin system questions

[zhangyin777]

Hello everyone

I would like to purify the newly synthesized protein by the biotin-streptavidin system, but in the process I have encountered some problems.

My strategy was to label the newly synthesized protein in vitro with biotin-puromycin, then bind the labeled protein with streptavidin-beads, and finally elute with 8M guanidine hydrochloride, pH 1.5. After I get the protein, I need to perform iTRAQ. analysis.

Question 1. When I detected the biotin-puromycin-labeled protein by western blot, I found that there are many proteins around 40kD. They may bind too many streptavidin-beads during the purification process, causing other proteins to fail to bind to beads, this may affect subsequent iTRAQ analysis. I wonder what these proteins might be? How do I eliminate or reduce the effect of these proteins on the purification process?

Question 2, My purification system is very inefficient, and the elution yields few proteins that can't satisfy the subsequent iTRAQ analysis. My current binding buffer and wash buffer are PBS. The elution buffer is 8M guanidine hydrochloride, pH 1.5, but why the protein detected in elution is very weak. What could be the reason?

Thank you very much!
 

[CharonY]

Biotin/strep purification almost always co-purifies some other proteins (I am not sure how precisely your synthesis/pulldown set up is so can't comment on that). To identify what you co-purified I would suggest to simply digest the sample and run it through an MS to identify them (i.e. a shotgun approach). At least that will give you an idea what you are dealing with.