(For a comprehensive listing of CRF’s see http://www.hiv.lanl.gov/content/sequence/HIV/CRFs/CRFs.html). By capturing A clade diversity, we capture some of the diversity found in the regions of CRF_02 that are A-like, but CRF_02 started with a recombinant founder

virus decades ago, and has been spreading and diversifying as a separate lineage (Zhang et al., 2010), and so it will have its own distinctive evolutionary trajectory. Each CRF represents its own lineage, thus by including the CRFs in diversity considerations, not just major clades, we take a more comprehensive and realistic view of global diversity than by a more narrow examination of major clades. Fig. 1B shows how many peptides were included in each clade- or CRF-specific peptide set (only sets that contain > 300 peptides are shown). If a peptide sequence was found in multiple clades/CRFs, then it was counted Buparlisib chemical structure in multiple sets. Peptides sets from the seven most frequent clades (A, B, C, D, G, CRF_01, and CRF_02) include > 500 peptides each. PepStar peptide microarrays were produced by JPT Peptide Technologies GmbH (Berlin, Germany). All peptides were synthesized on cellulose membranes using SPOT synthesis technology. Subsequent to a final synthesis step attaching a reactivity tag to each peptide’s N-terminus, the side chains were deprotected and the solid-phase bound peptides were

transferred into 96-well microtiter filtration plates (Millipore, Bedford, MA, USA). For cleaving see more the peptides from the cellulose membrane the individual spots were treated with aqueous triethylamine [2.5% (v/v)]. The peptide-containing solution was centrifuge-filtered into daughter plates and the solvent was removed by evaporation under reduced pressure. Quality control measurements using LCMS were performed on random samples of the final library. For transferring the peptides to 384 well plates, the dry peptide derivatives Tacrolimus (FK506) were dissolved in 35 μL of printing buffer and reformatted with automated liquid handling systems. Peptide microarrays were produced using a non-contact high performance microarray printer on epoxy-modified

slides (PolyAn; Germany). All peptides and controls were deposited in three identical sub-arrays, enabling analysis of assay homogeneity and reliability of the results. Peptide microarrays were scanned after printing process and statistical values were generated for identification and quality control of each individual spot. Subsequently, peptide microarray surfaces were deactivated using appropriate quenching solutions, washed with water and dried using microarray centrifuges. Resulting peptide microarrays were stored at 4 °C until use. Thirty-six (36) serum or plasma samples were obtained from previously performed studies in the Barouch laboratory and were selected to represent a spectrum of potential preclinical and clinical uses for the microarray.