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Blocking and Washing membrane - (May/13/2015 )

I am very new to Western blotting and I have been making some mistakes. My most recent mistake is that after blocking I did not wash again before primary antibody incubation. What is goingto happen to my membrane?


If you are not changing the protein in the buffer for Ab incubations, you are fine. If you use just buffer without milk/ BSA and save/reuse the Ab dilution then the Ab solution will not last that long.


Even if you did change the protein type, it should still be fine. The blocking step has little to do with the antibody incubation, it is primarily to coat the unused spaces (i.e. those areas outside the lanes where your proteins should be) on the membrane with protein.


as bob1 said, there should be no difference.


we don't wash after blocking. we only wash after antibody incubations.