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
My paper has all these details
Sc-30 santa cruz, 1:500; lysis buffer composition, WB procedure, loading dye composition and a novel oligomerization shift.
http://www.nature.com/articles/cddiscovery20163
Please let me know if you have any questions.
Good luck.
Dear Jinesh
Thanks a lot for your suggestion.I think most people recommend to use Santa Cruz Kras Ab. However, i tried initially with a little old stock of sc-30 maybe thats why it didn't work for me.
Hi,
Older K-Ras Sc-30 antibodies are good ones (concentrated than the newer ones, despite same clone). Antibodies stay good if properly stored at refrigerators, even for decades. K-Ras usually stays on membranes upon activation or inside nucleus or at mitochondria. These facts make it necessary that one has to do proper lysis to get it in Westerns. Try a longer 40 minute lysis as mentioned in the paper and use 15% gel or precast gradient gel (4-20%). Do not let the dye run out when running SDS-PAGE. Or try an immunofluorescence to see where it is located (protocol is in the same paper). Good Luck.
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