Libraries of overlapping peptides, generated based on the sequences of each protein in the WNV genome, were obtained through the Biodefense and Emerging Infections Research Resources Repository. These libraries consisted of overlapping peptides, typically 18–20 amino acids long with a 12 amino acid overlap (peptide length and overlap was adjusted as needed to avoid terminal cysteine residues). Peptides were dissolved in DMSO at 20 mg/ml and subsequently diluted as needed. Recombinant HLA-DR proteins were generated as described [38 (link)]. Briefly, each HLA-DR was purified from the supernatants of transfected insect cells, biotinylated, and dialyzed into 0.1M phosphate buffer. Biotinylated monomer was loaded with 0.2 mg/ml of peptide by incubating at 37°C for 72 hours in the presence of 2.5 mg/ml n-octyl-β-D-glucopyranoside and 1 mM Pefabloc SC protease inhibitor (Sigma-Aldrich, St. Louis, MO) and then conjugated using R-PE streptavidin (Biosource International, Camarillo, CA) at a molar ratio of 8 to 1.
R pe streptavidin
Manufactured by Thermo Fisher Scientific
R-PE streptavidin is a fluorescent conjugate used for the detection and quantification of biotinylated molecules in various applications such as flow cytometry, immunohistochemistry, and Western blotting. It consists of the streptavidin protein labeled with the R-phycoerythrin (R-PE) fluorescent dye.
Automatically generated - may contain errors
Lab products found in correlation
Pefabloc sc protease inhibitor, by Merck Group (3 mentions)
Pefabloc sc, by Merck Group (3 mentions)
N octyl β d glucopyranoside, by Merck Group (3 mentions)
High resolution hla dna typing kit, by Thermo Fisher Scientific (2 mentions)
Biotin ligase, by Avidity (2 mentions)
N dodecyl β maltoside, by Merck Group (2 mentions)
Streptavidin apc, by BD (1 mentions)
Streptavidin pe cf594, by BD (1 mentions)
Pefabloc, by Merck Group (1 mentions)
Pe cy5 streptavidin, by BD (1 mentions)
Cd86 gl 1, by BioLegend (1 mentions)
Mhcii m5 114, by BioLegend (1 mentions)
Streptavidin apc, by Thermo Fisher Scientific (1 mentions)
Facscanto 2, by BD (1 mentions)
Lsrfortessa, by BD (1 mentions)
7 aad, by BD (1 mentions)
Attune nxt instrument, by Thermo Fisher Scientific (1 mentions)
12 protocols using r pe streptavidin
1
Peptide Library Preparation and HLA-DR Binding
2
Production and Biotinylation of DRB4 Tetramers
3
Production and Tetramerization of Recombinant DQ8
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Recombinant DQ8 protein was produced as previously described [27] (link). Briefly, soluble DQ8 was purified from insect cell culture supernatants by affinity chromatography and dialyzed against citric/phosphate storage buffer, pH 5.4. For the preparation of HLA class II tetramers, DQ8 protein was in vivo biotinylated in Drosophila S2 cells [28] (link) prior to harvest and dialyzed into citric/phosphate buffer. The biotinylated monomer was loaded with 0.2 mg/ml of peptide by incubating at 37°C for 72 h in the presence of 0.2 mg/ml n-Dodecyl-β-maltoside and 1 mM Pefabloc SC (Sigma–Aldrich, St. Louis, MO). Peptide loaded monomers were subsequently conjugated into tetramers using R-PE streptavidin (Biosource International, Camarillo, CA) at a molar ratio of 8∶1.
4
Generation of HLA-DR Tetramers for Allele-Specific Analysis
5
HLA-DR Tetramer Generation and Binding
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Recombinant HLA‐DR proteins were generated as previously described.28 Briefly, each HLA‐DR was purified from the supernatants of transfected insect cells, biotinylated, and dialyzed into 0.1 mol/L phosphate buffer. Biotinylated monomer was loaded with 0.2 mg/mL of peptide by incubating at 37°C for 72 hours in the presence of 2.5 mg/mL n‐octyl‐b‐D‐glucopyranoside and 1 mmol/L Pefabloc SC protease inhibitor (Sigma‐Aldrich) and then conjugated using R‐PE streptavidin (Biosource International) at a molar ratio of 8 to 1. Fel d 1 tetramers were selected based on our prior studies.29 Specifically, a panel of 14 tetramers were used in this study; HLA‐DRB1*01:01 (2 tetramers), HLA‐DRB1*03:01 (3 tetramers), HLA‐DRB1*04:01 (1 tetramer), HLA‐DRB1*07:01 (1 tetramer), HLA‐DRB1*09:01 (3 tetramers), HLA‐DRB1*11:01 (1 tetramer), HLA‐DRB1*13:01 (1 tetramer), HLA‐DRB5*01:01 (2 tetramers). Each subject was typed for their expression of HLA‐DRB alleles using a high‐resolution HLA DNA typing kit (One Lambda). The appropriate tetramer(s) were selected based on the subjects’ expression of these HLA‐DRB alleles.
6
HLA-DRB1*04:01 Tetramer Preparation
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Recombinant HLA- DRB1*04:01 was purified from insect cell culture supernatants by affinity chromatography and dialyzed against phosphate buffer, pH 6.0. To prepare tetramers, DRB1*0401 was biotinylated in vitro and subsequently incubated with 0.2 mg/mL of peptide at 37°C for 72 h in the presence of 0.2% n-octyl-β-D-glucopyranoside (Sigma-Aldrich) and 1 mmol/L Pefabloc SC. Monomers were conjugated into tetramers using RPE streptavidin (Invitrogen) at a molar ratio of 8:1.
7
Recombinant DRB1*04:01 Protein Production and Peptide Loading
8
Recombinant DR0401 Protein Production
9
Protein Purification and Tetramerization
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DR0401 protein was purified from insect cell cultures as previously described58 (link),59 (link). Monomers were loaded with 0.2 mg/ml peptide at 37 °C for 72 h in the presence of 0.2 mg/ml n-Dodecyl-β-maltoside and 1 mM Pefabloc (Sigma–Aldrich). Peptide-loaded monomers were conjugated into tetramers using R-PE streptavidin (Invitrogen), PE-Cy5 streptavidin (BD), or PE-CF594 streptavidin at a molar ratio of 8:1.
10
Multiparameter Flow Cytometry Protocol
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Flow cytometry data was acquired using the BD FACS Canto II (Becton-Dickinson) or BD LSR Fortessa (Becton-Dickinson). Data analysis was performed using Flowjo (Treestar Software). Antibodies were purchased from BD Biosciences, Affymetrix/eBioscience and Biolegend. Antibodies for CCR7 (4B12), CD11b (M1/70), CD11c (N418), CD44 (IM7), CD115 (AFS98), F4/80 (BM8), Gr-1 (RB6-8C5), Ly-6C (HK1.4), Ly-6G (1A8-Ly6G), MHC I (34-1-2S), OX40L (RM134L), PD-1 (J43), PD-L1 (MIH5), PD-L2 (TY25) were purchased from eBioscience. CD25 (PC61), CD40 (3/23), CD80 (16-10A1), CD103 (M290), and annexin V antibodies were from BD Biosciences. 7AAD was also from BD Biosciences. CD86 (GL-1) and MHC II (M5/114.15.2) antibodies were from Biolegend. gp34-tetramer was prepared by sequential addition of streptavidin APC or streptavidin-R-PE (Invitrogen) to biotinylated gp34 monomers (H-2Kb; AVYNFATC) generously provided by the NIH Tetramer Core Facility.
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