Hi
Iam trying to make NIH3t3 mutant kras stable cells.For this, I have made kras(g12d) construct in pSIR neo vector. I infected nih3t3 cells using retrovirus and selected with neomycin for 5 days ( 1mg/ml). After having all uninfected cells die, I lyse my cells using RIPA buffer and check for protein exp using western. I do not see kras overexpression . I checked using CST kras12d antibody (D8H7), His Ab since my construct is His-kras.
But in 293T transfection of hisKras construct, i can detect kras using His Ab and not with CST kras12d Ab.
Can anyone suggest what might be happening ? Even after complete selection, I cannot detect kras in NIH3t3 cells . Pls share your suggesstions and protocols if any.
Also any advice on which Ras antibody can detect endogenous and exogenous kras in mouse fibroblast?
Thanks
Apoorva
Do you have a control cell extract in which you know for certain that your kras antibody is working? If not, that should be your first step.
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