Problem with nonspecific antibody binding

Khoo Keng Meng medp6023 at leonis.nus.sg
Fri Mar 7 23:32:37 EST 1997


Hello Homer,
	    Did you try blocking with a serum from the same species that
the secondary antibodies were raised in? e.g. goat serum to block
antibodies raised in goats. Alternatively, have you determined that
false-positive signal is not due to autofluorescence itself? Tissues tend
to autoflouresce in the blue-green region. Alternatively, you can try some
other fluorochrome like Texas Red or TRITC ...if it's auto flouresecence,
it shouldn't show up that much if you use there fluorochromes. If there's
still false-positive, you have to look into both your primary (don't
forget it could be your primary that's causing you all this trouble!) and
secondary again.
	    Hope this helps! 

Meng. 
Department of Biochemistry,
Faculty of Medicine,
National University of S'pore.

On 26 Feb 1997, DiverNLB wrote:

> Hello. I am currently developing a protocol for immunofluorescent
> detection of the stress proteins hsp70 and cpn60 on cryosectioned Mytilus
> mantle tissue. The problem I am having is nonspecific binding of the
> secondary antibody (conjugated to fluorescein), leading to strong
> false-positive signals. I have tried blocking with bovine serum albumin,
> fetal bovine serum, and horse serum, with little or no effect. Could
> anyone suggest any other solutions? Please respond to DiverNLB at aol.com.
> Thank you.
> 
> 
> Homer O. Ellison
> Dept. of Biological Sciences
> California State University, Long Beach
> 
> 
> 




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