Pierce antibody biotinylation kit for ip

Manufactured by Thermo Fisher Scientific

Sourced in Germany

The Pierce Antibody Biotinylation Kit for IP is a laboratory tool designed to covalently attach biotin to antibodies. This process enables the detection and study of antibody interactions during immunoprecipitation experiments.

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Lab products found in correlation

Pierce ms compatible magnetic ip kit, by Thermo Fisher Scientific (1 mentions) Streptavidin hrp, by Jackson ImmunoResearch (1 mentions) Tmb substrate, by Merck Group (1 mentions)

4 protocols using pierce antibody biotinylation kit for ip

Mass Spectrometry Analysis of SARS-CoV-2-S Immune Complexes

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For mass spectrometry analysis of SARS-CoV-2-S specific precipitates, individual sera containing FcγRIII/CD16-reactive soluble immune complexes were subjected to immune precipitation (IP) using Pierce MS-compatible magnetic IP kit (ThermoFisher Scientific, Darmstadt, Germany) according to manufacturer’s protocol. Briefly 250 µl serum was incubated overnight at 4 °C with 5 µg of biotinylated anti-RBD-specific TRES-1-224.2.19 mouse monoclonal antibody (1:20) or TRES-II-480 (isotype control; (1:80) (kind gift of H.M. Jäck, Erlangen) before addition of streptavidin magnetic beads. Beads were subsequently collected via centrifugation and elution buffer was added to detach putative precipitated antigen. The elution was dried in a speed vacuum concentrator and shortly run into 10% polyacrylamide gels. After over-night fixation (40% ethanol, 10% acetic acid, 50% water) and washing (3x), complete lanes were excised. Antibody biotinylation was performed using a Pierce antibody biotinylation Kit for IP (ThermoFisher Scientific, Darmstadt, Germany) according to manufacturer’s protocol.

Ankerhold J., Giese S., Kolb P., Maul-Pavicic A., Voll R.E., Göppert N., Ciminski K., Kreutz C., Lother A., Salzer U., Bildl W., Welsink T., Morgenthaler N.G., Grawitz A.B., Emmerich F., Steinmann D., Huzly D., Schwemmle M., Hengel H, & Falcone V. (2022). Circulating multimeric immune complexes contribute to immunopathology in COVID-19. Nature Communications, 13, 5654.

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Siglec-6 and FcεRIα Receptor Complexes

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Streptavidin complexes were created by mixing biotinylated anti-Siglec-6 (or isotype control) and anti-FcεRIα (or isotype control) mAbs at the indicated molar ratio (1:1, 3:1, or 9:1, anti-Siglec-6:anti-FcεRIα), then adding streptavidin at a 1:4 molar ratio and incubating at 4 °C overnight. Anti-Siglec-6 (clone 767329) was biotinylated using the Pierce Antibody Biotinylation Kit for IP (ThermoFisher Scientific). Biotin-conjugated anti-FcεRIα, mIgG2a isotype control mAb, and mIgG2b isotype control mAb were purchased from BioLegend. Complexes were produced such that the final treatment concentrations of anti-Siglec-6 and anti-FcεRIα in the 1:1 molar ratio complexes would equal 1.25 μg/mL, matching the concentrations of the free antibody treatments. HSMCs were incubated with streptavidin complexes or free antibody for 30 min at 37 °C before assessing cell activation and epitope blockade by flow cytometry.

Robida P.A., Rische C.H., Morgenstern N.B., Janarthanam R., Cao Y., Krier-Burris R.A., Korver W., Xu A., Luu T., Schanin J., Leung J., Rothenberg M.E., Wechsler J.B., Youngblood B.A., Bochner B.S, & O’Sullivan J.A. (2022). Functional and Phenotypic Characterization of Siglec-6 on Human Mast Cells. Cells, 11(7), 1138.

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Quantitative NS1 ELISA Protocol

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ELISA plates were coated with 5 μg/mL of capture mAb in 50 μL PBS/well and incubated at 4°C overnight. The next day, plates were washed once with PBS and blocked with PBS-BSA 3% (100 μL) and incubated for 1h at room temperature. The plate was then washed twice with PBS-T, and the serum or recombinant NS1 was diluted in PBS-BSA 1% (50 μL) before being added to the plate and incubated for 1h at room temperature. Plates were washed 4X with PBS-T, and the biotinylated detecting mAb (Pierce Antibody Biotinylation Kit for IP, ThermoFisher) was diluted in PBS-BSA 1% (50 μL) and added to the plate to incubate for 1h at room temperature. The plate was then washed 4X with PBS-T. HRP-streptavidin (Jackson Immuno) diluted in PBS-BSA (50 μL) was then added to the plate and incubated at room temperature for 1h. After incubation, the plate was washed 4X with PBS-T and 1X with PBS. The plate was then developed with TMB substrate (100 μL; Sigma) for ~15 minutes. The enzymatic reaction was interrupted using 2N H₂SO₄, and the plate was read at OD₄₅₀ nm. The concentrations of NS1 in sera were interpolated using a standard curve of recombinant NS1 ranging from 1 to 1000 ng/mL; the limit of detection for this assay was 2 ng/mL.

de Sousa F.T., Warnes C.M., Manuli E.R., Ng A., D’Elia Zanella L.G., Ho Y.L., Bhat S., Romano C.M., Beatty P.R., Biering S.B., Kallas E.G., Sabino E.C, & Harris E. (2023). Yellow fever disease severity and endothelial dysfunction are associated with elevated serum levels of viral NS1 protein and syndecan-1. medRxiv.

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Biotinylation of Anti-p27 Monoclonal Antibody

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The mouse anti-human p27 monoclonal antibody, mAb RSV7.10, was kindly provided by Dr. Gale Smith (Novavax, Gaithersburg, MD). The Pierce Antibody Biotinylation Kit for IP (Cat. # 90407, ThermoFisher Scientific, Waltham, MA) was used to biotinylate the mAb according to the manufacturer’s instructions with some modifications. Briefly, 100 μg of p27 mAb (100 μL of 1 mg/mL) was buffer exchanged in 1X PBS for compatibility with the biotinylation reaction. 100 μL of 1X PBS was then added directly to the kit-supplied NHS-PEG4-Biotin to a concentration of 8.5 mM. 3.14 μL of the biotin solution was then added to 96.84 μL of purified p27 mAb in a 1.5 mL microcentrifuge tube for a total of 100 μL per reaction. The mixture was gently rotated at 25°C in the dark for 1 h. Finally, the buffer was again exchanged to remove unbound biotin. The biotinylated p27 mAb stocks were stored in one-use aliquots at −80°C until use.

Blunck B.N., Aideyan L., Ye X., Avadhanula V., Ferlic-Stark L., Zechiedrich L., Gilbert B.E, & Piedra P.A. (2021). Antibody Responses of Healthy Adults to the p27 Peptide of Respiratory Syncytial Virus Fusion Protein. Vaccine, 40(3), 536-543.

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