From: clap@iastate.edu
Date: Wed Oct 13 1993 - 11:36:18 EST
I am interested in using antibodies to stain beta-galactosidase in lacZ transfected human melanoma cells. Specifically, I would like suggestions for fixation and permeabilization protocols that will retain this cytoplasmic protein within the cells but not denature the protein beyond antibody recognition. I intend to use flow cytometry to detect either Protein A-FITC or goat anti-mouse IgG-FITC "secondary antibodies." 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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