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Non-specific binding of protein in western blot - (Feb/16/2016 )

Hi guys,


There's some unspecific protein bands in my chemiluminescence developed pvdf membrane. I believe those protein bands are from the bacteria as they are not the right size, and appeared even in negative control. And there's few band shown on my protein ladder as well. My target protein band, however, is no where to be found.


Aren't only one band (my target protein) supposed to manifest as I am using the chemiluminescence treatment with specific primary antibody? 


I am confused.

-Meg P. Anula-

Yes... and no.


It is very very common for antibodies to bind non-specifically to other proteins on a blot. This non-specificity can be reduced by using an appropriate concentration of antibody (titrate it to balance signal with non-specific binding), using stringent conditions (e.g. higher salt concentration, more detergent(s)) for both the blocking/antibody incubation solution and washing steps. You can also use more washing steps.


Some antibodies are notorious for non-specific binding. I think BRACA1 has an antibody where you are supposed to cut off the bottom half of the membrane to remove an area where there are large amounts of non-specific binding, such that leaving the half on will drown out any specific signal.


I start with the minumum concentration as suggested by the antibody manufacturer.


If I have to re-determine the suitable concentration of my antibody, I should test the primary or the secondary or both?


What worried me now is that I couldn't see any specific signal also. T.T

-Meg P. Anula-

Do you try new loading buffer? It is possible that your sample has been polluted. 

How long is you blocking time? Try prolong the time.

Reduce your antibody dilution ratio.

-Andrea Fortina-