Multiple possibilities:

1) Did you  check if your protein is being really over-expressed before the affinity cromatography?

2) What exactly method did you use to obtain a stable FA fraction from PFA? Heating or increasing pH? Try to use commercial FA.

3) Have you tried methylene-blue or UV crosslinking methods?

Hi Alfonso,

               Thanks for responding! I checked the expression of my flag tagged protein both by doing a western and a reporter assay. It also responded to its inducer. The protein is expressed from its promoter on the chromosome.  But should it be overexpressed from a plasmid?

I made FA by heating PFA in PBS(pH7.2). I have not used methylene-blue or UV cross linking.

Silpi

Hi Silpi,

Probably you will get better result if you induce over-expression from plasmid, so , you will get more probabilities to catch that you are looking for. Additionally, you can try commercial FA in case your PFA treatment produce non-stable FA fractions. UV-based method is very useful for protein-nucleic acid.

Good luck

Thanks Alfonso! I will try both methods and let you know.