problem western help

ffr.de odin25 at netcom.com
Fri Feb 10 12:26:21 EST 1995


Recent western blot was stained prior to first antibody with amido black 
to visualize lanes so that they could be separated.  It was then blocked 
in milk followed by exposure to purified first antibody, diluted in the 
blocking buffer.  Upon washing a black precipitate formed on several 
lanes (but not others) which looked suspiciously like the amido black 
staining except that not all of the lanes were stained.  This occured 
with both bacterial cell lysates and mammalian cell lysates.  
Completeionj of the western still allowed normal AKP based detection to 
be used but I would like to hear theories as to the nature of the black 
staining during the wash after the first antibody.  Note: first antibody 
is simple Ig with nothing conjugated to it, wash buffer is standard 
TBS/tween.  We have checked for obvious problems in our buffers but have 
not found any.  This is not something which has been occuring previouly 
in our lab.  Though we will remake solutions I would still like to know 
what is causing this.  Thanks in advance for any help you can provide.



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