determination P-akt in buffycoat via Western blot

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poaske
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determination P-akt in buffycoat via Western blot

Hi,

we are trying to detect P-akt Ser473 in the buffycoat from blood from patients with cancer, treated with radiotherapy.
the isolation from the buffycoat has been done with histopaque. We did a western blot for P-akt Ser 473 from cell signaling and we had nice bands the first time we did the western blot. now we tried to repeat this but we do not get the same bands as we saw the first time, although we used exactly the same protocol as the first time.
Does anybody had this problem before?

gr