I've tried several times to do western blot of HIF1-alpha but failed. Has anyone managed to get good bands of HIF1-alpha under hypoxic condition using Abcam antibody (1:500) in a glove box? Can the protease inhibitor cocktail fully stop the degradation of HIF1-alpha? Thanks.
hi
you need check the correct antibody reactivity with positive control according to abcam company data sheet .Meanwhile, in western blot test the chance of getting the answer with a polyclonal antibody is greater than the monoclonal antibody.
"Lane 1 : MCF-7 (normoxia) Lane 2 : MCF-7 treated with 0.5% oxygen for 24 hours
Lysates/proteins at 30000 cells per lane."
good luck
HIF1a alpha is always difficult to work with. Please use a positive control like hypoxia exposed lysate (3% oxygen vs 20% oxygen). To me santa cruz antibody worked all.
Yes, it can in the ringt ration/concentration. You should try to find an adequate protocoll before performing your experiments. I woul suggest to use an antibody conc. of max. 1:1000.
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