p-Akt help!

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Haloperidol
Haloperidol's picture
p-Akt help!

I'm working with NCI-H322 cell line and trying to detect p-Akt by Western blotting with Cell Signaling Ser-473 p-Akt primary Ab and anti-mouse secondary.

The problem is that my p-Akt band keeps showing up at the wrong molecular weight. I work with another cell line (A549) and the p-Akt band shows up at the correct place (60-kDa) but H322 keeps giving me about 150 kDa bands for p-Akt even though A549 shows up fine in the same run.

Anyone got any ideas?

Thanks!

Guo-qiang Huang
Guo-qiang Huang's picture
Why don't you try Southern

Why don't you try Southern biotech's anti AKT antibody on NCI-H322 cell line . We have tried that antibody several times in different cell lines, it was constantly located on 57KD. You can call their customer service to get a free sample, the Catalog Number is 10946-01. the telephone number is 205-945-1774.

saswati1
saswati1's picture
you might want to try the

you might want to try the same antibody from a different company. I think the phosphorylated protein should show the band where it is supposed to show., So, with a different antibody and with a positive control ( here A549) , you might want to repeat the experiment.