The protein I am working on is a transcription factor and is abundant in nucleus. I want to do subcellular fractionation of the adherent tumor cells followed by ChIP sequencing in order to identify the downstream targets. I also want to perform co-immunoprecipitation followed by mass spectrometery of the nuclear fraction to identify the interacting partners of my protein. Kindly help me with the protocol which maintain the complex in native state and reduce the background for the downtream applications.
No one can recommend you a protocol that can maintain the complex in native state and reduce the background for the downtream applications.
This is because there is no knowing what protein you are interested in, what other proteins it interacts with, what buffer conditions maintain/violate their compositional integrity etc. These are the questions you have to find the answers yourself. Remember, when you are starting a new work, you are the leader! As Galadriel tells Frodo in Tolkien's LOTR, "If you don't find a way, no one will."
That said, you can start with a basic buffer composition, such as comprising Tris-Cl or Na/K-Hepes, ~20mM, pH ~7.5; salt NaCl/KCl around 150mM, EDTA ~1mM, some MgCl2 1-2mM; 5-20% glycerol, a detergent like Igepal/Triton at 0.2-1.0%, 1mM DTT and protease inhibitors.
This is a very non-intrusive composition, and should preserve many protein-protein interactions. That means, this buffer might also result in increased non-specific protein enrichment. Thus, you will have to incrementally try more and more stringent buffers to wash off the non-specific interactants (by increasing salt concn, detergent concn, changing detergent type to include triton, sod. deoxycholate, even mild SDS, etc.)
One more aspect I wish to emphasize:
In the literature, you see many people doing co-IP for transcription factors (TFs) from whole cell extracts (WCE) or nuclear extracts (NE), and implicating the interactions in transcription. For example, if A and B are both TFs and they co-IP from WCE/NE, people generally interpret them to be acting together in transcription. I strongly dislike this notion. Because, how many proteins and in what proportion and affinity interact in solution in the cytoplasm or nucleus has no meaning to transcription. To be relevant to transcription, the TFs have to be docked onto the chromatin. To cooperate/synergize in transcription, two TFs have to interact on the chromatin. So, for TFs, I prefer enriching the chromatin first, and then doing the immunoprecipitation from the soluble chromatin as the starting material (there are many easy ways to get to the chromatin). This is chromatin-IP-mass-spec/Western is incredibly problematic, though! Because, two TFs might appear to interact with each other even if they are tens of nanometers apart and are actually bridged by the chromatin/DNA. So, it is important that the chromatin/DNA is destroyed using a non-specific nuclease, such as Benzonase (again, hoping that the two do not fall off after the DNA is gone).
The experiments that you are planning are very common experiments these days, and a good few days' self-study will give you plenty of guidance. What you may seek help with is in troubleshooting down the road. But I hope you don't have to!
Good luck.
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