I'm doing research on protein (A and B isoform) that form enzymatically active hexameric complex. These hexamers are represented in all combinations (A6, A5B,..., B6). I would like to find out if they are all equally represented in my cell line or some hexamers are preferred? I can do immunoprecipitation and after that I tried native electrophoresis (without SDS) and western blot, but didn't get any results. Do you have any idea what should I do after ip in order to separate and distinguish these hexamers?
Hi Martina, if you have antibodies to these isoforms you could try doing native page without IP and see which ones show up after stripping the blot and probing with other antibodies. Alternatively you could try ion exchange chromatography to separate out the various hexamers and blot them.
If all else fails, you could try treating them with cleavable crosslinkers like https://www.thermofisher.com/order/catalog/product/22586 IP them with RIPA buffer, cleave them with BME and blot them.
Isoelectric focusing may be able to provide a useful degree of separation if theisoforms have different pIs. Going further, 2-D electrophoresis would increase the resolution if the two isoforms have significantly different molecular weights.
The issue you describe is a nanoscopy problem, in my opinion. If you have access to cryoelectron microscopy (cryoEM) or atomic force microscopy (AFM), those will be good bets. GL.
I humbly disagree with Adam (since IEF is very slow and may promote dissociation of the hexamers; on the other hand, 2D-PAGE will certainly dissociate the hexamers). I also disagree with Vincente (for sure, AFM is not going to distinguish between untagged hexamers of different compositions) and am puzzled by Jai's claims, since your hexamers must be quite large and I am not sure they would fly well in MS.. Overall, I think Steingrimur's suggestions maybe a good sttarting point, particularly with reference to the reversible crosslinkers.
I suggest the circular dichroism to distinguish ration of different types of coupled monomer as best way. I think 2D-Page is insensitive method and MS-MS is best way to see which combination is in here, but it is hard to say to ratio. Best way is circular dichroism to determine secondary structure of protein, so you can see all combinations and their ratio very easly by changing of dissociation conditions.
Perhaps a combination of chemical crosslinking to prevent subunit dissociation, and IEF for separation would work.
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