Hello everyone, I am working on a project for hiv 1 integrase. I have performed cloning and according my seq results it was successful. It has been few weeks I’m trying to detect my protein on western but it doesn’t show up. Hiv 1 integrase is getting degraded so I am using proteasome inhibitor(mg132). I have tried several treatments but none of them has worked. I did 50ul for 3 and 4 hours, 20ul for 4 hours, 10ul for 8hours and 6hours, 2ul for 18hours. I seed the cells and thennafter 24 hours I make the transfection with my pcmv5 flag integrase plasmid and then after 24 hours I start the treatments. Cell line used is Hek cellsZ any advice is valuable. Thank you
Hello Panagiota,
I would like to help out but there's a lot of information concerning the work that you're doing that is missing
Could you send a message with the detailed steps you went through during your experiment so that I can see what could the problem be ?
I think your treatment time is very lengthy, MG132 treatment should not be exceeding more than 6 hrs otherwise cells would undergo apoptosis. You need to be careful with concentration as well. I feel if you are over expressing in HEK cells ,first try detecting your over expression is working or not by probing with anti Flag antibody.
First you can try with the dotblot .......if it works then go for westrenblot................................
I think the epitope is not available to bind by mAb
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