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

Hi,
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.

-luciana-

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.

-bob1-

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.

-luciana-