Hi all,

I am overexpressing mutant kras(g12d) in NIH3t3 cells and selecting with Neomycin (my construct is kras(g12d) in pLJM-linker with Neo res gene). After complete selection when uninfected cells die, I prepare lysates of stable NIH3t3 cells but unable to detect Ras overexpression using santa-cruz Ras Antibody (1:100) 

I am unable to detect even the basal ras levels. Can anyone please suggest a good Ras antibody with their personal experience or relevant papers that they can share.

Also, what is the best way of preparing western lysates for Ras detection?.  

I pellet the 3t3 cells, wash pellet with PBS then lyse the pellet using RIPA buffer. I centrifuge at high speed, isolate the supernatant and mix with 2X loading dye , boil for 5 mins then use this as sample for SDS PAGE.

Thanks

Apoorva