After performing the co-IP, repeatedly I see that the protein in the final elution fraction run at different molecular weight (slightly lower for the co-precipitated and higher for the target protein) as compared to that in the crude lysate and unbound fractions run side-by-side on the same gel. The samples are denatured and boiled as usual for SDS-PAGE. The antibody used is very specific to the protein of interest. Is it possible that during co-IP performed overnight at 4degC, the protein is modified? Any comment or suggestions will be appreciated.
Show the gel please. It will dramatically increase the number of answers, also flag your question with familiar/popular topics Biotechnology/Biochemistry. Please...
Sorry, but at this moment this is only help I can offer;)
I had several incidences where precipitated proteins started forming some sort of covalent conjugations which could not be broken up by adding SDS, Beta-mercaptanol, or boiling. I think this is because increasing local concentrations of proteins by trapping them on beads could increase the probability that these proximal proteins react with each other in vitro. Likewise, during the IP reaction, dephosphorylation or other type of covalent modification could be possible unless you inhibit the enzymes.
- Badges
- Science method