Western Using Monoclonals

[wang qi]

Dear netters,

I am have some trouble with  western blotting using monoclonal 
antibodies.

Several monoclonal antibodies have been generated in our lab by injecting 
mice with whole cells. One of our antibodies recognizes an antigen on the 
cell surface using indirect-immunoflouresence microscopy. We suspect that  
the antigen is a membrane-associated protein because the cells are labeled 
after they are treated with sugar-digesting enzymes but not with 
proteases.  We, however, have not been able to identify any specific band 
on the immunoblots. The condition we used is similar to that for 
polyclonal antibodies. We tried different BLOTTO's such as BSA/TBST, 
BSA/TBS, Milk/TBST, Milk/TBS. Noting worked.

Thanks in advance for any comments and suggestions.

QiWang
Dept. of Biological Sciences
The Univ. of Iowa
qiwang at blue.weeg.uiowa.edu