So, the problem happening here is when I am doing blots to check GFP in my cell lysates, bands are occuring at higher molecular weight then the actual GFP protein has i.e bands are developing above 55KDa but the mol weight of GFP is around 25KDa. This is the case when I am using 5% milk buffer in TBST for blocking.
But when I used 3% BSA in 1XPBS for blocking bands developed at 2 sites- one is around 25KDa and other is above it around 35/40KDa. But the blots are not looking clear when I am using BSA.
Anyone can tell why is this happening?
Is there protein interaction happens when you use GFP with milk buffer?
And how can I troubleshoot this issue?
Hi,
you mean you have two bands within one sample when using BSA? One at around 25kDa and one at 35/40kDa? Or in different samples?
In my experience BSA leads to more unspecific binding than blocking with milk.
In case it is within one sample then I would trust the BSA more with the band around 25 kDa and the one above is possibly a non-specific signal. Do a proper control without GFP transfection and maybe also try blotting with secondary antibody only without primary antibody detecting GFP, because secondary antibodies can lead to unspecific signals as well.
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