Western smear

soenke behrends behrends at plexus.uke.uni-hamburg.de
Wed Jun 14 03:26:43 EST 2000


Dear netters, 

we have raised different antibodies to different
proteins using KLH coupled peptides. While most
antibodies work fine and cause no problems, we
have a peculiar smear problem with two different
antibodies raised against different epitopes of 
the same protein. The phenomenon appears as a dark
band-like signal at 70 kDa. From there we get 
sometimes a little punctuated  streaks to the top
and bottom of the gel. We can also discern our 
protein as a nice strong band, but the whole blot looks 
rather shitty. We have then used a fancy Boehringer
blocking reagent and a 1:1000 antibody dilution 
(instead of 1:500). With this we got rid of the 
smear effect, but the signal from our protein was
rather weak now. What I do not understand: 
Why do we get this effect with two different antibodies
raised against different epitopes of the same protein?
With the antibodies raised against the other proteins
we do not have any such problems. 
Maybe pure coincidence?

any hint or tip is greatly appreciated

Thanks for your time and help
Soenke Behrends












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