Hi, I just did a quick and dirty preliminary experiment to look for the surface expression of an inducible recombinant protein in E. coli and got a very puzzling result. I am staining with polyclonal antisera raised in rabbits to the native form of the recombinant bacterial protein that I know works in an ELISA. The secondary is a commercial goat anti-rabbit IgG-PE. I didn't titrate the antibodies, so I expected a certain amount of background. In the induced culture, I found that the entire population shifted nicely to the right when the primary and secondary antibodies were present compared to the autofluorescence tube. However, the secondary antibody alone resulted in 2 populations. One, matching the autofluorescence peak and the other (roughly 50% of the sample) staining significantly brighter than the positive (primary + secondary). In the uninduced culture, there was little staining in either the primary+secondary tube or in the secondary alone tube. Both the induced and the uninduced came from the same culture which was split and incubated in the presence or absence of arabinose for 4h. I would appreciate any insight the you may have regarding these results. Cheers, Wallly Wallace Lauzon, PhD Dept. Biochemistry, Microbiology, and Immunology Faculty of Medicine University of Ottawa 451 Smyth Rd Ottawa, Ontario CANADA K1H 8M5 Phone: (613) 562-5800 x8244 FAX: (613) 562-5452
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