I am currently working with mTOR inhibitors and assessing their effect through the phosphorylation of S6K, but apparently the antibodies I am using are not specific enough.
I am attaching an image of my western blot. I am using drug concentrations above the IC50 for inhibition of mTOR, so phosphorylation of S6K in Thr389 should be inhibited. However, I am getting signal in all of my samples and a change in molecular weight for the band of the untreated sample. Any ideas why is this happening? Can anyone share their experience with antibodies for phosphorylated S6K?
Thanks Franck Morceau
! I am also using S6K1 Ab, and as you said, the inhibition of P-S6K1 with the treatments can be observed. However, it is difficult to do quantification of these bands when they are so close together. For that reason I am using specific phosphorylated S6K1 Ab.Thanks again for your response!
What company's antibody are you using. I just published a paper as a co-author using the phospho-p70 S6 kinase (thr389) from cell signaling (cat # 9205) and it worked really well. Also, what molecular weight are you getting in your blots? I saw the image, but there was no ladder to assess what the different molecular weights you are getting. Also, for your samples, did you normalize for total protein with a BCA assay or run a house keeping gene? Unequal loading could account for some differences.
thanks for your response Brandon A Kemp ! I am using antibodies from cell signaling (cat # 9205 ) as well. I am getting ~65 kDa and ~70 kDa bands. I used BCA assay for protein normalization and I am also using actin AC15 antibodies to evaluate the loading. There was no variation on the signal for the actin bands, indicating fair loading for every sample.
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