Hi everybody, this is my first question in this forum! Excuse my english please!
I have been getting problems with my western blots. The blots are very very dirty. The high background is due to the use of SuperSignal Femto from Pierce. I am using a very low amount of protein amb trying to check some MAPK phosphorilated. I have been using primary antibodies from Cell Signal and my 2aries antibodies are from Dako and BD Bioscience. In my test everything is ok but I have no signal in my PVDF mb. Then i decided to use SuperSignal Femto, the most sensitive, and I have a good bands but an imposible background. I have tryed everything:
-different buffers: PBS, TBS, TBS from Cell Signal...
-different membranes: PVDF, nitrocellulose
-different blockade buffers: milk 5%, milk 10%, milk 5%+ BSA 2%, milk 5%+Goat or rabbit serum 2%, milk 10%+1%BSA, milk 10%+1%BSA+1% normal serum...
-different shacking rotors..
Absolutly everything. The best results was obteined using the milk10%+1%BSA blocking buffer, but now the background is higher again.
I am really getting crazy... I need desperately some help...I need this approach to continue with my PhD project and my supervisor is requiring me results... I am very anguished
thanks a lot for your help