Phospho proteins

[letisolari]

Hi! I am having problems trying to do a WB with c-kit (should give a band of 110kD)and phospho c kit I used b actin as control, b actin worked very nicely but the others didn't. I used red ag and anti oxidant. Could it be the transfer? I did coomasie and I saw more bands than usual in the gel...or the antibodies? Any suggestion?

[admin]

hey Letisolari,
Sometimes its a problem with the antibody, however I can tell you there are tricks for phospho-protein western blots and one of them is loading the entire cell lysate directly with SDS-loading buffer.

Did you see any bands with your antibody or faint ones? Also a good idea would be to use phosphatase inhibitors and protease inhibitors such as PMA. Did you use phosphatase inhibitors?

Can you post your gel here to see??

cheers

[letisolari]

thanks for the suggestions. I used Na fluoride( phosphatase inhibitor), Na orthovanadate (phosphatase inhibitor), Aprotinin(protease inhibitor), Leupeptin (protease inhibitor), Pepstatin (protease inhibitor), PMSF (protease inhibitor), EDTA (calcium chelant). I got two bands, neither of them at the right molecular weight, and some light background. I will post the WB tomorrow.

you suggested me loading the complete lysate on the gel?

thanks again