chemiluminescent immunodetection

Zhongguo Xiong zxiong at
Wed Feb 2 10:55:51 EST 1994

In article <94029.135153MSH4 at> Peggy Halleck <MSH4 at> writes:

>Hi!  We have been using  chemiluminescent detection of HRP-labelled secondary a
>ntibodies or protein A on immunoblots and sometimes have problems with very str
>ong signals appearing as white spots or "negative staining" on the film.  Is th
>is due to quenching and does anybody know how (short of starting over and reduc
>ing the amount of antigen on the blot or increasing the dilution of the antibod
>ies involved) this can be fixed when it happens?

I had some experiences with the chemiluminescent Western blot and saw similar 
things described here. It was "reverse blot" in my terms. The problem can be 
cure easily by using a reduced amount of primary and secondary antibodies.  
You need to experiment with different dilutions to find the best combinations.

On the other side, I never had any luck with chemiluminescent wester. I used 
antibodies to a plant virus encoded protein to probe the synthesis of the 
protein in plant cells and always end up with several bands that are not 
expected sizes. The same bands also appeared in plant extracts that was ot 
infected by the virus. I had several antibodies to different proteins. The 
same bands showed up no matter what antibodies I used. I suspected that some 
of the plant proteins have endogenous HRP activity, but a control with no 
added antibodies did not show anything. 

I'll be delighted to hear any comments, interpretation, or similar experience.


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