I am attempting to detect differences in Akt phosphorylation at Serine 473 with Cell Signaling Technology product #9271 (Rabbit anti-phospho-Akt).
Typical lysis conditions and standard SDS-PAGE, wet transfer were used. Phosphatase inhibitors were present in the lysis buffer. I did not perform immunoprecipitation, but rather loaded a total tissue lysate. I loaded approximately 50ug total protein on the gel.
Using an antibody against total Akt (not just the phosphorylated form), I see a band of the expected size.
However, I could not detect any signal with the phospho-Akt antibody.
Since the control antibody made by the same company worked, I can surmise that 1) the upstream techniques worked and 2) I definitely have Akt in my protein samples.
So, why am I not detecting phospho-Akt? Is phosphorylation quickly lost during lysis? Inadequate phosphatase inhibitor concentration?
Any help would be much appreciated!
I´ve used exactly the same antibody without any problems, I always have seen a really good signal. Using milk for blocking...weird.
Hello Damian,
Considering that phosphorylation events are transient, and that phosphorylated proteins comprise a relatively small portion of the total protein, you might just not have enough pAkt (S473) to begin with.
In addition, working with tissue as opposed to cell-only samples could "hide" your phosphorylated protein in a sea of unknown and virtually uncontrollable factors as well.
All the best,
Shahin
I recommend that you do a positive control either in the same cells or in COS-7 cells (not HEK cells) stimulated for 2-5 min with EGF 100 ng/ml. Compare starved to EGF- stimulated cells.
Good luck !
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