Hello everyone, hope you are doing well.
I am doing the co-IP assay recently. My aim is analyse the interaction of protein A and protein B. Importantly, protein A can be glycosylation. I constructed plasmid of A tagged with flag, as well as B tagged with HA. I transduced the plasmids into 293T cells and followed the protocol of co-IP. I tried different immunoprecipitation, firstly, I IP with flag-beads and IB with HA. The result is good and specific. However, when I IP with HA-beads and IB with flag, I found the bound is not the same molecular weight as input samples.
My target protein A is about 36kD-40kD, and change to 55kD after glycosylation by WB assay. The IP results showed the protein A reduced its molecular weight to 36-40kD. I wondered if the co-IP pull down the un-glycosylation protein? I have repeat for 2 times and got the same result which make me puzzled.
Hope someone can give me any suggestion.
Thank you.
Hi there,
It might be that glycosylation of A doesn't resist to coIP process. Did you actually probe your flag pull down with flag antibody? It would tell you about the behavior of A protein during the process (comparing the profile of input and output).
Hi Lee,
it might well be that A only interacts with B when unglycosylated. However, there are different other possibilities, like glyco-loss as dominique stated, or even that you pull down truncated forms.
Is your overexpressed protein A-FLAG gylcosylated like the endogenous protein A (Same molecular mass?)
If you can get enough CoIP-ed protein A, you could cut out the band and use mass spectrometry to investigate protein and glycoslyation state.
Best
Christian
are you including protease inhibitors in your lysis, IP, and wash buffers?
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