The gene X has been taken from host namely Z, cloned and expressed X in E.coli. I would like to understand the interaction partners of X protein in host A. As X is his-tagged protein, purified and kept with whole cell protein of host Z along with Ni-NTA for binding and eluted out. I could see only Protein A in the gel, not any interacting partner from host Z
Is there any other way to find the unknown partner?
Your reply would be greatly appreciated
Hi Dhanashree,
The best method in my opinion is to start with Immunoprecipitation of the whole host proteins using antibody against X .
N-NTA chromatography may alter X-host candidate protein(s) interaction.
Best wishes
If I understand your question correctly, you want to incubate bacterially expressed his tagged protein in cell lysate and find the interacting partners of that protein after Ni-affinity chromatography. In principle you should see the interacting partners after Ni-affinity since bound proteins elute along with your His tagged protein.
But experimentally you may have to optimize several parameters like protein concentration of your his tagged protein as well as cell lysate. The idea is that if the interacting proteins are very low in concentration, they may not find access to your histagged protein and moreover you may see nonspecific binding. Adjust the concentration to ideal. If you see several bands on SDS gel after Ni-affinity, better to find the identity of those proteins and try individually.
Buffer optimization is also important since you do not speculate the kind of interactions(ionic, hydrophobic). If the salt concentration is high for the interactions, you may probably disrupt the ionic interactions. Also pay attention to salt concentration in wash buffers during Ni affinity purification.
As the above person suggested immunoprecipitation is sensitive than Ni affinity if a good antibody is available.
Patrick Rouimi AND Vidhyadhar Nandana
Thank you for your valuable suggestion I'll just look into it
Do you expect your interaction partner(s) to be membrane proteins? If so, you will need to add a detergent to your lysate to solubilise the membrane proteins. I would also recommend crosslinking to improve the stability of any complexes formed.
Check first if co-immunoprecipitation works. You can use also life-time imaging microscopy.
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