blocking and primary antibody in different buffers? - (Jan/19/2012 )
I did western of cytokine by blocking the filter in BSA 5%, primary and secondary in diluted in 5% BSA too. I developped wit hECL plus, I got very huged background all over the film. no signal at all.
I decided to wash the filter in PBS-T 0.1% for while and reblock the filter in 5% milk instead og BSA.
I used the same aliqot of primary antibody diluted in 5% BSA overnight as previous western, and secondary in milk 5%.
DO you think its feasible and eventually could see my band in the film??
Or I should change something else??
It should be fine, though you may find that you still have high backgroung unless you quenched the HRP from your first attempt.