I am currently working on Co-IP of NRIP and MyoD by overexpressing both proteins in HEK293T cell. The question is I cannot get a specific band of MyoD. For my input, I think the protein level is too low to be detected so I will increase the protein amount to run a western blot. However, several bands appear when detected using MyoD antibody after co-IP. The band most likely MyoD is pointed with a blue arrow. Some of my colleagues say it may be endogenous MyoD or protein with similar conformation. Does anyone face a similar situation when working with this protein? or any reference paper suggested for studying this protein. Thank you.
Dear Hui Yee Ng
That is a common issue of Co-IP, IP, or pull-down assay. I suggest to match the corresponding antibody's kDa with the position marker.
Article Western Blot: Technique, Theory and Trouble Shooting
https://elifesciences.org/articles/80140
Kind regards
AB Bayazid
I am not really working with this specific protein, but I would think different phosphorilation states of your protein or degradation of your protein.
the extra band are the light (25kd) and heavy (50kd) chains of the antibody you used for IP: there is several way to at least dimished these bands: use secondary antibodies from different species (and with low cross-reactivity), use (anti GFP) antibody cross-linked to beads, use non-reducing laemmli buffer (if your myoD does not have disulfide bonds (none reduced Igg will run at 150kd) ....
Hello Hui Yee you could also try to use PICO to detect your MyoD. They claim to be much more sensitive compared to Co-IP/WesternBlot workflows. Here is an explanation of the technology:
https://www.youtube.com/watch?v=ptZx-IgLW3I
- Badges
- Science topic