chemiluminescent immunodetection

John R Barta jbarta at
Sun Feb 6 12:09:58 EST 1994

Zhongguo Xiong (zxiong at wrote:
: 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.

: Xiong

I have been using AP-based chemiluminescent westerns and have had little
trouble with the blots.  The "white" areas are apparently the result of
the localized consumption of the chemiluminescent substrate in areas of
high enzyme concentration.  You can check this by comparing blots obtained
immediately after adding substrate with those obtained after a significant
incubation (30 minutes plus).  The un/underexposed areas should be larger
in the latter blots.  The solution to the problem (lower concentrations of
Ab's) have been mentioned earlier in the thread.


J. R. Barta			e-mail: jbarta at
Dept. of Pathology		voice: 519-824-4120 ext. 4017
Ontario Veterinary College
University of Guelph		"Usual Disclaimers Apply"
Guelph, Ontario

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