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how to remove aspecifics on western blot filter - (Mar/16/2011 )

Im pissed off about western blots!
I did several western blots, but, recently, idont succeed to have clean western blot in terms of signal of my protein.
today, I ran a gel, fresh running buffer, fresh transfer buffer, the wet transfer was good, I checked by ponceau staining, then, i blocked in 5%milk-PBS tween 0.1%. later, I incubate the filter with actin antibody,mouse, Sigma, 1:10 000 for 1 h, and secondary anti mouse 1:5000 for 1h.
I got good signal on actin molecular weight, but, unfortunantly, I have many aspecifics in the filter in the middle and upper part of the filter! also, some lanes were saturated!
I develop with ECL plus, and using the STROM scanner to visualise my western.
Actually, Im not convinced about this method, since, I was using in the past, kodak film to develop, I was successful.
But, now with this storm, I have very often saturation bands!,
on the other hand, I have to use the same filter to do western blot of another protein around 55kda. Im afraid to have hidden signal due to these aspecifics and i could not quantify later!!! I could strip also before, but, i dont like stripping, since I could lose proteins and not homegen lanes!!
Please if you have any advices for troublshooting and understanding better the origin of these aspecifics, thanks in advance.


Often this sort of background is caused by too high an antibody concentration, usually the secondary. You can also try incubating for less time with the secondary and/or diluting the primary further.

Sometimes antibody degradation can also cause these effects, if you don't aliquot and store the antibody at -20, you probably should, despite the data sheet telling you to store at 4 deg C.


thanks for your reply!
in fact, the primary antibody is stored at -20, secondary is aliqotted at 4c. since, its not the first time I use it, with the same problem
of saturation and aspecifics! may be its degradated.
Also, the concentration of secondary 1to 5000 is probably high, for HRP- anti mouse.
I alo suppose that the milk I use is not good anymore, old!! Im not sure if the milk could induce these aspecifics. but, I usually use milk instead of BSA, I have good signals.