Question
How to stabilize a protein-protein interaction in a Far-Western Blot?
I have been testing the interaction of two proteins from a parasite involved in homologous recombination, that is already been proved in humans but so far I haven't seen anything at all. Apparently this is a weak interaction so I'm afraid I've been losing it when I wash the membrane so I decided to use glutaraldehyde as a cross-linking agent but I have no idea what concentration to use or if it is a good agent to use. Can you tell me the concentration to use or another idea to stabilize the complex?
All Answers (7)
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It would be helpful to know if you were testing for the interaction in crude extracts or were looking at purified samples of bait and prey molecules. Having looked at the above mentioned paper, I noticed that Far Western blotting is not very specific as a screening tool. Immunoprecipitation either of non denatured cell extracts or of crosslinked extracts seems to be a bit more specific. This would work even better if there was a physiological or chemical treatment which would be expected to increase or decrease the interaction in vivo.
-D -
First of all, thanks for your comments. I'm testing purified samples and I have also tried the same assay denaturing and renaturing the protein on the membrane and then probing it against the prey protein, but still no signal. -
How are your controls? Do you have a good positive control for the antibodies you are using? I expect you are using an antibodies made against homologous proteins do they perform as expected in a regular Western taken through the crosslinking protocol?
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As Douglas has suggested, could you provide more detail about your protocol from start to finish (including buffers used for lysis/washing, epitope tag used on bait protein (if any), antibody used for IP)? This may help us identify potential problems with the co-IP. Also, have you tried just doing a traditional western blot, assuming there are good antibodies for your proteins of interest?
Just a quick suggestion about the crosslinking step - I would recommend using a crosslinker which is thiol-cleavable so that when you do SDS-PAGE, the cross-linked complex will dissociate and run at their expected individual sizes. I have used DSP (from thermo scientific, http://www.piercenet.com/browse.cfm?fldID=02030234) with success. -
What antibodies are you using? Potentially, your antibody might bind your protein on the surface that interact with your other protein hence dissociating the complex. Have these specific antibodies been used for co-IP before?
Good luck! -
I am having just that problem with a N terminal GFP tagged protein. I have been using Nano Trap beads( Camelid anti-GFP beads) to coimunoprecipitate a complex (which I know is there). I don't see it but I see another protein that interacts with the GFP protein at a different place. The place where the failed interaction occurs is on the N terminus of the GFP labeled protein and I think the GFP binding by the Nano Trap beads interferes with the interaction. One thing that might would is to isolate the complex on gel filtration column The column fractions could be assayed either by elisa or Western blotting- I plan to do this in my case.
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