help:need info. on primary/secondary binding

SHANNON DUNN g1400111 at nickel.laurentian.ca
Thu Aug 17 10:47:16 EST 1995


Hi, 

I've been having problems detecting an isoform of myosin heavy chain protein
(which I know for sure is present in the tissue) using an indirect method. My
 primary antibody is mouse IgM and the secondary is a peroxidase-conjugated 
goat anti-mouse IgM. I performed an ELISA and found that there is a problem 
with the primary and secondary binding. The secondary is working well with 
other IgM primary antibodies and the primary antibody is freshly purified from
a hybridoma. Can anyone suggest some reasons why this problem is occuring?
Furthermore, I have recently read a paper which used these same anti-MHC
antibodies. They used Tris(hydroxymethyl)aminomethane-buffered saline
for  the IgM antibodies and phosphate-buffered saline for the IgGs.
Could anyone suggest why two different buffers were used? 

Thanks,

Shannon Dunn
Neuromuscular Research Group, Laurentian University
g1400111 at nickel.laurentian.ca




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