hi,
I am working with GFP tagged connexin 36 transfected in hela cells. I do a crude membrane preparation and then used chaps to solubliize the membrane for two hrs and run it on a 10% gel. When I probe with GFP antibody, Im getting a band only for GFP which is very predominant, but not for GFP connexin 36. Tried all the gimmicks, nothing seem to work. Is anyone out there faced similar situation with the same or different proteins. Suggestions are welcomed
Thanks
jaya
Hello Jaya. If you detect the GFP, the immunoblotting is working. Do you include proteinase inhibitors in your homogenization and solubilization buffers? Maybe you could also try a much more rapid protocol just to validate. After you isolate your crude membrane preparation, suspend it up buffer containing 1-2% triton X-100 using a Dounce or Potter Elvehjem homogenizer (15-20 passes, on ice). Centrifuge. Run the supernatant on SDS-PAGE/immunoblot. Resuspended the pellet in 1X Laemmli buffer, heat, and run it as well. Keep all your buffers cold and work on ice or in a cold room. If you don't detect the full length fusion protein, recheck your plasmid.
Bruce
Thanks for the answer. Iam using protease inhibitors in the lysis buffer and the same buffer which I use to resuspend the membrane pellet. Since this is a membrane protein and Iam trying to standardise for Co-IP, I did not try Triton X-100 rather tried CHAPS and beta malto pyranoside. My guess is that the protein and GFP are cleaved in some way, but I dont know how to prove it.
Thanks
jaya
Do you have an antibody to the other part of the fusion protein? Is it present in your extracts?
The problem is there arent any good antibody for connexin 36, that is why we fused it to GFP
Hi Jaya,
I think your protein is cleaved from GFP-tag and you could find it by protein molecular weight on western blotting. Of course you must have 2 bands in WB if it cleaves partially.
That is the problem. I get a band around 28 KDA which I know it's for GFP, but I see nothing for the GFP with protein which should be around 70
if it forms dimer, it might be better to use reduced form or the MW would increase in which 10% acrylamide is too concentrated for it. your protein can not penetrate to gel easily.
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