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. 2014 Feb 4;53(8):1302–1310. doi: 10.1021/bi401289p

Figure 1.

Figure 1

ELISA analysis of binding and photo-cross-linking of azF-CXCR4 mutants to 12G5 mAb. (a) CXCR4 schematic showing sites of azF incorporation. (b) Binding signal normalized to total expression, which is detected in permeabilized HEK293T cells using the 1D4 antibody to a C-terminal epitope. (c) Experimental scheme for photo-cross-linking and detection. HEK293T cells expressing the mutants were incubated with 12G5 and then exposed to UV light. After being washed with a low-pH, high-salt buffer, the cells were incubated with the HRP-coupled anti-mouse secondary antibody. The complexes were detected by HRP-catalyzed formation of a fluorescent product from Amplex Red. (d) Relative photo-cross-linking level obtained by normalizing to the signal from Y184azF-CXCR4. Fluorescence emission at 590 nm was detected with excitation at 530 nm in a multiwell fluorescence plate reader instrument (CytoFluor II, PerSeptive Biosystems). Error bars in panels b and d represent the standard error of the mean from three independent trials, each performed in duplicate or triplicate.