ECL Western Blotting, need help

Yankiwski yankiwski at aol.com
Thu Aug 7 23:19:26 EST 1997


I would like to thank all of you for suggestions and ideas regarding my
Western blots.  
Perhaps if I address some questions that arose I can get more helpful
feedback.

NaN3 seems to inhibit the activity of HRP coupled to my secondary
antibody.  Elimination of azide from my solutions yielded more consistent
results;  it appears that extended storage of my Ab in 5% dry milk in TBS
preserved with azide inactivated the HRP, so re-use of the Ab resulted in
no exposures.  

My primary antibody is affinity purified, twice.  The first purification
is over Sepharose conjugated to all proteins except my antigen.  This
supernantant is then passed over an affinity column coupled to the Ag, to
yield a pure rabbit polyclonal.  When this is used in my Westerns I get
several bands.  Background is not very high, indicating that my washes are
quite good, but how can I eliminate this nonspecific binding?  Maybe
producing a monoclonal is the only way.  Different concentrations of
primary and secondary have been tried, as have secondary anti-rabbit Abs
produced by goat and donkey.

Perhaps the binding of the Ab IS specific:   if my epitope is highly
charged, isn't it possible that the Ab is binding other charged epitopes
of various proteins?  Also, maybe what I'm seeing is binding to a degraded
form of my antigen, and although a variety of protease inhibitors are
used, some degradation probably still does occur. 

Sorry for the long post, but thanks once again for the helpful thoughts.

Victor



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