The conditions that failed to solubilize the receptor (blue and pink data points) cluster at zero. platforms, which limits the size of the analyte to larger molecules (>5000 Da). Overall, the two approaches proved highly complementary: both assays identified identical detergents that produced active solubilized CCR5, as well as those detergents that either were ineffective solubilizers or inactivated the receptor. Keywords:array, Biacore, CCR5, Flexchip, GPCR, optical biosensor, surface plasmon resonance == Introduction == Surface plasmon resonance (SPR) biosensors can be used to identify suitable conditions for isolating G-protein-coupled receptors (GPCRs) from membranes while preserving the receptors abilities to bind conformationally sensitive ligands. For example, we have used SPR in the past to screen GPCR solubilization, purification, and crystallization conditions [13]. We identified components of solubilization and crystallization buffers that increased a GPCRs stability and we optimized affinity-purification methods to yield highly active receptor. Here we outline two biosensor-based assays that reveal how different detergents affect the yield and activity of solubilized GPCRs. We used CCR5 as the model system since we have access to high-quality reagents and binding partners. Our CCR5 construct is usually C-terminally tagged with a peptide sequence recognized by 1D4 Alloepipregnanolone antibody [4], which stably captures the receptor yet can be regenerated at the end of each binding cycle. To probe the conformation of CCR5, we used a Fab of the 2D7 antibody. The detergent screening methods were developed using Biacore 2000 and Flexchip platforms. The microfluidics system in the Biacore 2000 can test one analyte across a reference and up to Alloepipregnanolone three reaction surfaces [5]. After each binding cycle the surfaces are regenerated and new aliquots of receptor are captured. While serial sample processing decreases throughput, this devices sensitivity (it is routinely used to examine molecules <500 Da), combined with its ability to monitor both ligand capture and analyte binding, makes it useful for receptor screening. In the Biacore Flexchip array platform, an analyte is usually flowed across a matrix of ligand spots within a single large-format flow cell which can significantly improve ligand throughput [5,7]. But array biosensors have lower sensitivity than traditional serial platforms; Flexchips limit of detection is usually approximately 5000 Da. And, at present ligands are spotted off line so capture levels, for example, cannot be directly quantitated. Our screening methods took advantage of the unique features of these two platforms. With Biacore 2000 we established the correlation between ligand capture and Fab binding levels. For Biacore Flexchip we devised a rapid assay that should prove Mouse monoclonal to IgG2a Isotype Control.This can be used as a mouse IgG2a isotype control in flow cytometry and other applications particularly useful for GPCRs that are less stable than our CCR5 construct. Using either approach, we could bin the detergents into three groups: those that did not solubilize CCR5, those that solubilized but inactivated the receptor, and those that yielded active solubilized receptor. == Materials and Methods == == Materials == The Biacore 2000 and Flexchip optical biosensors, as well as instrument-specific consumables (e.g., sensor chips, affinity chip kits, amine-coupling kits, and standard 96-well plates and covers), were obtained from GE Healthcare/Biacore (Uppsala, Sweden). Cf2Th canine thymocyte cells overexpressing the human chemokine receptor CCR5 tagged with a C-terminal linear peptide tag (C9: TETSQVAPA) were propagated by the National Cell Culture Center. Purified mouse anti-human CD195 (CCR5) monoclonal antibody 2D7 was provided by the National Institutes of Health AIDS Research and Reference Reagent Program and purchased from BD Biosciences (San Jose, CA, USA); the C9 tag-recognizing antibody 1D4 was purchased from the University of British Columbia (Technology Transfer Office, University of British Columbia, Vancouver, British Columbia, Canada). Detergents were purchased from Anatrace (Maumee, OH, USA), DOPC:DOPS (7:3) lipid mixture from Avanti Polar Lipids (Alabaster, AL, USA), polyethylene glycol (PEG) Alloepipregnanolone 8000 from Promega Corp. (Milwaukee, WI, USA), ImmunoPureFab Preparation Kit from Pierce (Rockford, IL, USA), and protease inhibitor tablets from Roche Diagnostics (Indianapolis, IN, USA). All other reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA) and Fisher Scientific (Rockford, IL, USA). == Preparation of 2D7 Fab == One mL of.