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So far, formaldehyde fixation (1.8% for 15 min. @ 37 C) followed by Triton
X-100 permeabilization has resulted in little or no specific binding of my
mouse anti-BG IgG. Likewise, a simple fix and perm in 90% methanol gave
unsatifactory results.
I have used BSA in Trition, Blotto/Tween20, and Blotto/Tween20 + 20% Normal
Goat Serum as blocking agents. Each resulted in similarly low background
binding. Unfortunately, the specific binding does not appear. However,
overall fluorescense is increased in the presence of primary antibody.
Thus, it appears that the primary is binding non-specifically and the secondary
is binding to the primary. Therefore, I don't think the problem lies in high
background obscurring the specific binding. Rather, it seems that specific
binding of the primary antibody to beta-galactosidase is absent. Help!
I want to use this technique to distinguish between transfected and
untransfected cell populations in mixed cultures so that each can be
analyzed individually for scatter, DNA, and possibly other intracellular
or surface antigens. Any suggestions would be greatly appreciated.
Thanks.
Jeff Clapper
Cell Facility, Iowa State University, Ames, Iowa 50011
(place mail text here)
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