Hi everyone!
I'm new here, I have trouble identifying the interaction between protein A and its potential partners.
Took a year doing all kinds of purficacion and I can not find proteins that may be forming a new complex with my protein of interest.
I made several Tadem Affinity purifications, and every time out different proteins that ended up being false positives.
I tried to change the conditions of lysate, incubation with resins and antibodies, but we have no positive result yet.
Is that the story is more complex still, my protein of interest "A" has a weight of 320kda and conditions for removing them are a little confusing, we have tried to use CHAPS, or a lysis buffer containing 10% glycerol, and we without finding any group of proteins that then we can verify that actually interact.
In this process we are already 4 protein that has been ruled out by co-inmunoprecipitations, immunofluorescence, placing different types of taps (protein C, TAP, etc.). And we have seen in some cases to get the IP positive in only one direction, pulling my protein "A", we get the protein "partner"but when we pulled the partner does not get to see my protein "A" ..
I understand it is hard to understand my situation, but if you have any advice on how to improve specific joints in this type of purification, or protein complex to see more specifically, would be infinitely grateful to you.
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