Blots are representative of at least two indie trials. Expression of azF-CCR5 Mutants We were interested in comparing the epitope maps of mAbs 2D79 and PRO 14010 in the AMG-510 CCR5 EC2 loop. and BzF-substituted CCR5 in complex with maraviroc.41 These results agree well with structural studies that indicate that the requirement is in the range of 2C4 ?.44 Here, we describe a microplate-based detection strategy, with potential for high throughput, which is based on targeted loss-of-function mutagenesis and subsequent photo-cross-linking using genetically encoded UAAs to study antibodyCreceptor complexes. The method relies on a sensitive cell-based enzyme-linked immunosorbent assay (ELISA) to detect fluorimetrically the transiently bound or photo-cross-linked mAb. We used the strategy to map complexes between 12G5 and CXCR4, with a focus on the role of residues in EC2. We also produced maps that depict the contribution of residues in EC2 on CCR5 for binding of mAbs 2D7 and PRO 140. In our method, we describe two parallel assays: one that identifies loss-of-binding azF mutants and another that identifies photo-cross-linked AMG-510 residues. In essence, we use the same mutants to identify and confirm the primary hot spot of conversation, as well as proximal sites that may be not indispensable for binding yet allow the formation of a stable covalent adduct. This is the first description, to the best of our knowledge, of whole cell detection of photo-cross-linked mAbCGPCR complexes. Our targeted mutagenesis and photo-cross-linking approach should provide a general framework for mapping any GPCR epitope. Materials and Methods Materials Antibodies were obtained from the following sources: 12G5 (eBioscience, catalog no. 14-9999), 2D7 (BD Pharmingen, catalog no. 555990), T21/8 (eBioscience, catalog no. 14-1957), PRO 140 (gift from J. P. Moore at Weill Cornell Medical College), 1D4 (National Cell Culture Center), and horseradish peroxidase (HRP)-labeled goat anti-mouse (KPL, catalog no. 474-1806) and goat anti-human (Jackson Immuno Research, catalog no. 709-036-149). Protein A/G UltraLink was purchased from Pierce (catalog no. 53132), and for 3 min. The cell pellets were solubilized for 1 h on a nutator at 4 C in a buffer made up of 1.5% (w/v) for 10 min at 4 C, and the supernatant fraction was AMG-510 treated with NuPAGE-LDS gel loading buffer (Invitrogen), supplemented with 100 mM DTT. The samples were then loaded on 4 to 12% Bis-Tris gels (Invitrogen) and electrophoresed in MOPS gel running buffer. After the proteins in the gel had been AMG-510 transferred to a PVDF membrane (Millipore, catalog no. IPVH00010) at 18 V for 45 min using a semidry transfer apparatus (Bio-Rad), the membrane was GRIA3 blocked in TBS-T [10 mM Tris-HCl buffer (pH 7.4), 150 mM NaCl, and 0.05% (v/v) Tween 20] supplemented with 5% (w/v) nonfat dry milk for 1 h at RT. The membranes were then incubated with 0.5 g/mL 1D4 antibody in PBS supplemented with 0.5% (w/v) BSA (PB buffer) overnight at 4 C. The next day the membrane was washed extensively in TBS-T, followed by incubation with the HRP-coupled goat anti-mouse antibody diluted 1:20000 in TBS-T supplemented with 5% (w/v) milk for 1 h at RT. Following TBS-T washes as explained above, the protein bands were revealed with enhanced chemiluminescence detection reagents (Pierce) and detected with HyBlot CL autoradiography AMG-510 film (Denville Scientific). ELISA-Based Detection of Photo-Cross-Linked Samples After main antibody incubation, the plates were placed on a chilly pack and exposed to 365 nm UV light (Spectroline Maxima ML-3500S) for 15 min at 4 C at a distance of 3 in. from the source. Subsequently, the wells were washed twice, each time with 150 L of 50 mM glycine-HCl buffer (pH 2.5) supplemented with 500 mM NaCl (G500 buffer). Then the wells were washed once with Pc/mB, followed by secondary antibody incubation as explained above. Western Blot Detection of Photo-Cross-Linked Samples After the cells in PBS had been harvested in the absence of protease inhibitors, the pellet was resuspended in PB buffer made up of the appropriate conformation-dependent.