I am checking effect of my compound on breast cancer cells, i ran western blot, i am attaching photo, and i have following questions-
1- i have not used phosphatase inhibitor in sample preparation but still get signals,how to explain this.
2- i am getting two adjacent bands one dark and one light , why ?
3- why i have not got band in sample lane 4, although on membrane with ponceu staining prior to antibody exposure showed high expression of protein???
Thanks in advance.
To be able to help you with your observations, please label your left panel with bands for protein size markers and tell me you used which antibody to detect proteins. Lack of a phosphatase inhibitor may not have serious consequence but did you add protease inhibitors?
Hello sir,
I am using E labscience Biotech,NFKB-p65 Monoclonal Antibody.
Yes i used protease inhibitor cocktail during cell lysis.
I think this is total nfkapa b p65 , i measured.
And for phosphorylated ( activated nfkappa b), i need to use both phosphatase inhibitor, and phospho nfkapa b antibody, this is what i understand till yet please correct me if i am wrong. Lane 1,2,3,4 is treated with drug and lane 5 is breast cancer cell without drug.
And i was reading the mechanism of nfkappa b family, but its little complicated to understand as there are canonical , noncanonical pathways, so i am goin through literature.
Sir please also tell me, how i can strip pvdf or store it if cant continue process at any step.
And thank you so much for reply.
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