I'm repeating my experiments for three times. They all end up with the same problem. I can't detect the protein X with mass spectra. Please help me confirm whether it's the antibody issue. The purpose is to IP protein X and identify the interactive protein with X via LCMSMS. The procedure was: 1. IP X from the cell lysate; 2. elute protein from beads with loading buffer (4xNuPAGE LDS sample buffer+0.1M DTT+RIPA lysis buffer);3. run the SDS gel; 4. stain with coomassie blue; 5. cut the band in gel and go with in-gel digestion; 6. inject sample into LC-MALDI-TOF/TOF. Theoretically, I should see X and other proteins with mass detector since I'm doing IP for X. Why does not it show up from the mass spectra? Besides, I did check the X with western blot before and after IP. They look fine and the negative control is fine too. I use mouse primary antibody for X from Santa Cruz. X is a nuclear protein.
I have a few questions/suggestions on your post. Do you detect other proteins in the mass spec? If you are seeing other proteins I would have to agree you are having an antibody issue or a false positive. Lastly, how intense are your [Only registered users see links. ] bands? Are they well defined and intense? If they are faint bands you may have a mass spec detection issue.
Laboratory Technician at Protea Biosciences, Inc. [Only registered users see links. ]
Thanks for your post. Refer to your question, I did detect other proteins (not my interest) including those contaminations from MSMS. The band from coomassie blue staining is well defined and intense when I upscale the starting cell lysate. Now I'm testing another antibody from different species and try on beads digestion. Hopefully it brings some good news. Please let me know if there's anything else I can improve. Thanks!