Biotinylated monomers of HLA-DQA1*0301 and HLA-DQB1*0302 were produced in-house using previously published protocols with either portion of the insulin B chain (Ins12–20 or Ins13–21) or the insulin connecting peptide (CP65–75, CP59–70). All biotinylated monomers were purified in two steps by NTA affinity chromatography followed by gel filtration on a Sephacryl-300 HR column (Cytiva). Aliquots were flash frozen and kept at −80°C until use. To tetramerize, the monomers were incubated with streptavidin-phycoerythrin (SA-PE) (Invitrogen), overnight at room temperature at a 5:1 ratio of monomer to SA-PE. This formulation was diluted to 1X via FW and used directly on cells. c. I-Ag7 monomers were tetramerized as described above with Ins12–20 and Ins13–21 and 9Q (a mimotope for the BDC2.5 antigen) monomers. Both I-Ag7 and HLA-DQ8 tetramers were diluted down to 1X with FW prior to staining cells.
Streptavidin phycoerythrin sa pe
Manufactured by Thermo Fisher Scientific
Sourced in United States
Streptavidin-phycoerythrin (SA-PE) is a conjugate molecule composed of the protein streptavidin coupled to the fluorescent dye phycoerythrin. SA-PE is commonly used in various biomedical and life science applications that require the detection and visualization of biotinylated targets.
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Lab products found in correlation
Sarkosyl, by Merck Group (1 mentions)
Tetramethylammonium chloride, by Merck Group (1 mentions)
Bio plex 200, by Bio-Rad (1 mentions)
Coelenterazine h, by Promega (1 mentions)
Dulbecco s modified eagle medium, by Thermo Fisher Scientific (1 mentions)
Anti ha antibody, by Cell Signaling Technology (1 mentions)
Opti mem 1, by Thermo Fisher Scientific (1 mentions)
Hepes buffered phenol red free medium, by Thermo Fisher Scientific (1 mentions)
Isopropyl beta d thiogalactopyranoside iptg, by Promega (1 mentions)
Anti myc antibody, by Cell Signaling Technology (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Anti ha agarose, by Thermo Fisher Scientific (1 mentions)
Apelin 12, by Phoenix Pharmaceuticals (1 mentions)
Human umbilical vein endothelial cells (huvec), by American Type Culture Collection (1 mentions)
Chinese hamster ovary cells, by American Type Culture Collection (1 mentions)
Bio plex 200 instrument, by Bio-Rad (1 mentions)
5 protocols using streptavidin phycoerythrin sa pe
1
Biotinylated HLA-DQ Tetramer Preparation
2
Multiplex Virus Detection via Oligonucleotide Coupling
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Oligonucleotide coupling; thirteen capture probes were included in the multiplex assay (Table1 ). Each probe sequence represented the reverse complement to the target region of the biotinylated PCR product. Different sets of carboxylated fluorescent microbeads were obtained from Luminex Corp. (‘s-Hertogenbosch, The Netherlands), and oligonucleotide probes for target viruses were assigned to individual bead sets. The oligonucleotide coupling was done according to the manufacturer’s instructions (xMAP cookbook, Luminex). The probe-coupled beads were counted using a hemocytometer and were stored in the dark at +4 °C. Hybridization; probe-beads and PCR products were hybridized as published [53 (link)] except that the streptavidin-phycoerythrin (SAPE, Invitrogen) incubation temperature was 48 °C. After three washes the amplicons were labeled with 4 μg/mL SAPE conjugate in 2 M tetramethylammonium chloride (Sigma), 75 mM Tris, 6 mM EDTA and 1.5 g7 L sarkosyl (Sigma), pH 8.0; for 20 min in the dark. Measurement; after three washes, the beads and the SAPE signal were analyzed in a Bio-Plex 200 (Bio-Rad).
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Oligonucleotide coupling; thirteen capture probes were included in the multiplex assay (Table
3
Apelin Receptor Characterization in CHO and HUVEC Cells
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Chinese hamster ovary cells and human umbilical vein endothelial cells were obtained from American Type Culture Collection (ATCC). All restriction enzymes were from NewEngland BioLabs. [Ala13] apelin-13, apelin-12, 13, 15, 17, 36 were purchased from Phoenix Pharmaceuticals. Lipofectamine 2000, streptavidin-phycoerythrin (SA-PE) and Opti-MEM I were obtained from Invitrogen Life Technologies. Isopropyl-beta-D-thiogalactopyranoside (IPTG) and Coelenterazine h were obtained from Promega. HEPES-buffered phenol red−free medium, Dulbecco’s modified eagle medium and Incomplete RPMI-1640 culture medium were purchased from Gibco. Anti-HA-agarose was obtained from Pierce Chemical Co. Polyclonal horseradish peroxidase-conjugated goat anti-rabbit immuno-globulins/HRP was obtained from Zhong Shan Gold Bridge Biology Corporation (China). Anti-Myc antibody and anti-HA antibody were purchased from Cell Signaling Technology
4
Luminex-based HPV L1 Protein Binding Assay
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HPV16 and HPV18 binding was determined by a Luminex-based assay utilizing glutathione-S-transferase (GST)-HPV L1 fusion proteins that were coupled with magnetic beads, as previously described (Katzenellenbogen et al., 2015 (link)). In brief, GST, GST- HPV L1 (16, 18) and GST-Protein A (subcloned from a plasmid kindly provided by Dr. Alice Prince, Columbia University) fusion proteins were expressed from a modified pGex4T vector (Katzenellenbogen et al., 2015 (link)), and crude bacterial lysates prepared. Magnetic microspheres (beads) containing a unique combination of fluorescent dyes (Bio-Rad Laboratories, Hercules, CA) were covalently coupled with glutathione-linked casein. Modified beads were used to purify fusion proteins from the crude lysates with each bead set incubated with a different fusion protein. Beads were washed, mixed together and incubated with culture supernatants in 96-well plates (final dilution 1:10). Antibodies that bound to the fusion proteins were detected by incubation with biotinylated anti-human IgG or IgM (KPL, Gaithersburg, MD) followed by streptavidin-phycoerythrin (SAPE) (Invitrogen). Fluorescence was measured on a BioPlex 200 instrument (Bio-Rad Laboratories). The median fluorescent intensity (MFI) for GST was subtracted from the MFI of the other antigens. GST-protein A was included to detect the presence of any IgG in the culture supernatants.
5
Multiplex Quantification of Environmental Pollutants
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Estradiol(E 2 ), bisphenol A(BPA) and [N-Morpholino] ethanesulfonic acid (MES) were purchased from Sangon Biotechnology Co., Ltd. (Shanghai, China). Polychlorinated biphenyls(PCBs), profenofos, atrazine(Atz) were purchased from Beijing Putian Tongchuang Biotechnology Co., Ltd. Different uorescence-encoded MagPlex® MBs with carboxyl group were purchased from Luminex Corp. (Austin, TX, USA). Aptamer and biotin-cDNA purchased with Shanghai Shenggong Biological Company (Table S1) and dilute to 10µM before use. Streptavidin phycoerythrin (SA-PE, 1 mg/mL) was supplied by Invitrogen Corp. (Carlsbad, CA, USA). 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride. (EDC), N-Hydroxy-sulfo-succinimide (S-NHS) were purchased from Pierce, USA. All reagents are analytical grade.
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