For phosphospecific kinase proetin how much time incubation (45') for secondary antibody is good for perfect result and washing time after incubation?

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I am using BSA for blocking membrane for Phosphospecific antibodies but getting much background, could any one help me how to overcome this problem?

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I am working on CHK2 kinase protein and every times after western blot getting blackground stain also, washed 3 times10 minutes each,what is problem ?

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Dennis Alexander Agardy

I always incubate with secondary for 1 hour at room temperature on a shaking table. After the secondary I wash 3 times for 10 minutes with TBS-T. Some other people prefer to wash only 3 times for 5 minutes. Try both and see what's working better for you, but I guess it would not make a big difference.

Tylen Chen

I agree with Dennis Agardy, 1h for secondary antibody incubation at RT is enough for most WB tests. In addtion, as for wash time, I think it depends on your result. If the background is high and the interesting band have showed up, just prolong the washing time (10minX3)or add the washing tmes(5minX5). Just have a try ,then optimize the procedure as needed.