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Fsl biotin

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FSL-biotin is a lab equipment product manufactured by Merck Group. It serves as a biotinylation reagent used for the covalent attachment of biotin to target molecules.

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Lab products found in correlation

High protein binding 96 well elisa plates, by Corning (4 mentions) 1 steptm ultra tmb elisa substrate solution, by Thermo Fisher Scientific (3 mentions) Piercetm high sensitivity streptavidin horseradish peroxidase hrp conjugate, by Thermo Fisher Scientific (3 mentions) Wallac 1420 victor2tm, by Agilent Technologies (3 mentions) Cytation 5 cell imaging, by Agilent Technologies (3 mentions) Gen5 microplate reader and imager software, by Agilent Technologies (3 mentions) 4 nitrophenyl α d galactopyranoside pnpg, by Merck Group (1 mentions) Purified porcine gb3, by Matreya (1 mentions) 18 0peg2000pe, by Avanti Polar Lipids (1 mentions) To horseradish peroxidase, by Merck Group (1 mentions) Anti flag antibody, by Merck Group (1 mentions) E64 inhibitor, by Peptide Institute (1 mentions) Recombinant human cathepsin l, by R&D Systems (1 mentions) Sucrose, by Carl Roth (1 mentions) 1 2 dioleoyl sn glycero 3 phosphocholine, by Avanti Polar Lipids (1 mentions) Bovine serum albumin (bsa), by Thermo Fisher Scientific (1 mentions) Streptavidin hrp conjugate, by Thermo Fisher Scientific (1 mentions) Human igg1, by Merck Group (1 mentions) Hrp conjugated anti flag antibody, by Merck Group (1 mentions) Ultra tmb substrate, by Thermo Fisher Scientific (1 mentions)

12 protocols using fsl biotin

1

Competition Capture ELISA for sEC1-2 Mutants

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The capacity of sEC1-2 mutants to compete with the binding of sEC1-2(WT) to rVSVs, bearing ANDV or SNV Gn/Gc, was determined by a competition capture ELISA. High-protein binding 96-well ELISA plates (Corning) were coated with purified sEC1-2 (100 ng/well) overnight at 4 °C, washed briefly with PBS, and blocked with 5% nonfat dry milk in PBS (1 h at RT). Pre-titrated amounts of rVSV-ANDV-Gn/Gc and rVSV-SNV-Gn/Gc, were membrane-labeled with a short-chain phospholipid probe, functional-component spacer diacyl lipid conjugated to biotin (FSL-biotin (5 µg/mL); Sigma-Aldrich) for 1 h at 37 °C. The rVSVs were pre-incubated with serial 2x- or 3x-diluted sEC1-2(WT or variants) for 1 h at RT prior to their incubation with sEC1-2 coated wells (1 h at 37 °C). Bound rVSVs were detected by incubation with PierceTM High Sensitivity Streptavidin-horseradish peroxidase (HRP) conjugate (1:10,000 dilution, Thermo Scientific). ELISA signal was developed using 1-StepTM Ultra TMB-ELISA substrate solution (Thermo Scientific) and measured at an absorbance at 450 nm on a Perkin Elmer Wallac 1420 Victor2TM microplate reader or Cytation5 cell imaging multi-mode reader (Agilent BioTek Gen5 Microplate Reader and Imager software, V.3.2).
Slough M.M., Li R., Herbert A.S., Lasso G., Kuehne A.I., Monticelli S.R., Bakken R.R., Liu Y., Ghosh A., Moreau A.M., Zeng X., Rey F.A., Guardado-Calvo P., Almo S.C., Dye J.M., Jangra R.K., Wang Z, & Chandran K. (2023). Two point mutations in protocadherin-1 disrupt hantavirus recognition and afford protection against lethal infection. Nature Communications, 14, 4454.
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2

Glycolipids and Lectins Interaction Study

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1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (referred to as 18:0 PEG2000 PE), and cholesterol were purchased from Avanti Polar Lipids; Bodipy FL C5-HPC and Texas Red 1,2-dihexadecanoyl-sn-glycero-3-phosphaethanolamine (DHPE-TxRed) from Life Technologies; and purified porcine Gb3 from Matreya. FSL-Lea(tri) referred to as DOPE-Lea(tri), FSL-biotin referred to as DOPE-biotin, 4-nitrophenyl-α-D-galactopyranoside (PNPG), and ethylenediaminetetraacetic acid (EDTA) were obtained from Sigma-Aldrich. The Cholesterol-Substituted Glycopeptide with a GalNAc-moiety (CSG-peptide)32 (link) was received from Prof. Klas Ola Blixt (University of Copenhagen). Recombinant LecA and LecB were produced from Escherichia coli following previously published procedures27 (link),63 (link). VVL was purchased from Vector Laboratories; recombinant Shiga toxin 1 B-subunit (StxB) from Sigma-Aldrich.
Villringer S., Madl J., Sych T., Manner C., Imberty A, & Römer W. (2018). Lectin-mediated protocell crosslinking to mimic cell-cell junctions and adhesion. Scientific Reports, 8, 1932.
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3

Biotinylation and Capture of rVSV-EBOV Particles

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The viral lipid envelopes of rVSV-EBOV GP particles were labeled with biotin using a function-spacer-lipid construct (FSL-biotin) (Sigma-Aldrich) for 1 hr at pH 7.5 and 37°C, as described (Ng et al., 2014 ). Biotinylated viral particles bearing GPCL were generated by incubation with thermolysin, and then captured onto high-binding 96-well ELISA plates precoated with recombinant streptavidin (0.65 μg/mL; Sigma-Aldrich). Plates were then blocked with PBSA, and incubated with serial dilutions of test mAbs. Washed plates were then incubated with a pre-titrated concentration of soluble, FLAG epitope-tagged, NPC1 domain C (NPC1–C) protein (Bornholdt et al., 2016a (link)), and bound NPC1–C was detected with an anti-FLAG antibody conjugated to horseradish peroxidase (Sigma-Aldrich). All incubations were performed for 1 hr at 37°C.
Wec A.Z., Herbert A.S., Murin C.D., Nyakatura E.K., Abelson D.M., Fels J.M., He S., James R.M., de La Vega M.A., Zhu W., Bakken R.R., Goodwin E., Turner H.L., Jangra R.K., Zeitlin L., Qiu X., Lai J.R., Walker L.M., Ward A.B., Dye J.M., Chandran K, & Bornholdt Z.A. (2017). Antibodies from a Human Survivor Define Sites of Vulnerability for Broad Protection against Ebolaviruses. Cell, 169(5), 878-890.e15.
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4

Competition Capture ELISA for sEC1-2 Mutants

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The capacity of sEC1-2 mutants to compete with the binding of sEC1-2(WT) to rVSVs, bearing ANDV or SNV Gn/Gc, was determined by a competition capture ELISA. High-protein binding 96-well ELISA plates (Corning) were coated with purified sEC1-2 (100 ng/well) overnight at 4 °C, washed briefly with PBS, and blocked with 5% nonfat dry milk in PBS (1 h at RT). Pre-titrated amounts of rVSV-ANDV-Gn/Gc and rVSV-SNV-Gn/Gc, were membrane-labeled with a short-chain phospholipid probe, functional-component spacer diacyl lipid conjugated to biotin (FSL-biotin (5 µg/mL); Sigma-Aldrich) for 1 h at 37 °C. The rVSVs were pre-incubated with serial 2x- or 3x-diluted sEC1-2(WT or variants) for 1 h at RT prior to their incubation with sEC1-2 coated wells (1 h at 37 °C). Bound rVSVs were detected by incubation with PierceTM High Sensitivity Streptavidin-horseradish peroxidase (HRP) conjugate (1:10,000 dilution, Thermo Scientific). ELISA signal was developed using 1-StepTM Ultra TMB-ELISA substrate solution (Thermo Scientific) and measured at an absorbance at 450 nm on a Perkin Elmer Wallac 1420 Victor2TM microplate reader or Cytation5 cell imaging multi-mode reader (Agilent BioTek Gen5 Microplate Reader and Imager software, V.3.2).
Slough M.M., Li R., Herbert A.S., Lasso G., Kuehne A.I., Monticelli S.R., Bakken R.R., Liu Y., Ghosh A., Moreau A.M., Zeng X., Rey F.A., Guardado-Calvo P., Almo S.C., Dye J.M., Jangra R.K., Wang Z, & Chandran K. (2023). Two point mutations in protocadherin-1 disrupt hantavirus recognition and afford protection against lethal infection. Nature Communications, 14, 4454.
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5

Competition Capture ELISA for sEC1-2 Mutants

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The capacity of sEC1-2 mutants to compete with the binding of sEC1-2(WT) to rVSVs, bearing ANDV or SNV Gn/Gc, was determined by a competition capture ELISA. High-protein binding 96-well ELISA plates (Corning) were coated with purified sEC1-2 (100 ng/well) overnight at 4 °C, washed briefly with PBS, and blocked with 5% nonfat dry milk in PBS (1 h at RT). Pre-titrated amounts of rVSV-ANDV-Gn/Gc and rVSV-SNV-Gn/Gc, were membrane-labeled with a short-chain phospholipid probe, functional-component spacer diacyl lipid conjugated to biotin (FSL-biotin (5 µg/mL); Sigma-Aldrich) for 1 h at 37 °C. The rVSVs were pre-incubated with serial 2x- or 3x-diluted sEC1-2(WT or variants) for 1 h at RT prior to their incubation with sEC1-2 coated wells (1 h at 37 °C). Bound rVSVs were detected by incubation with PierceTM High Sensitivity Streptavidin-horseradish peroxidase (HRP) conjugate (1:10,000 dilution, Thermo Scientific). ELISA signal was developed using 1-StepTM Ultra TMB-ELISA substrate solution (Thermo Scientific) and measured at an absorbance at 450 nm on a Perkin Elmer Wallac 1420 Victor2TM microplate reader or Cytation5 cell imaging multi-mode reader (Agilent BioTek Gen5 Microplate Reader and Imager software, V.3.2).
Slough M.M., Li R., Herbert A.S., Lasso G., Kuehne A.I., Monticelli S.R., Bakken R.R., Liu Y., Ghosh A., Moreau A.M., Zeng X., Rey F.A., Guardado-Calvo P., Almo S.C., Dye J.M., Jangra R.K., Wang Z, & Chandran K. (2023). Two point mutations in protocadherin-1 disrupt hantavirus recognition and afford protection against lethal infection. Nature Communications, 14, 4454.
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6

Quantifying Antibody-Mediated NPC1 Binding

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Thermolysin cleaved VSV-GP (rVSV-GPCL) was incubated with a functional-spacer-lipid reagent conjugated to biotin (FSL-biotin, Sigma) to allow incorporation into viral membrane, and the resulting biotinylated viral particles were then captured onto streptavidin coated plates, as described previously (Ng et al., 2014 ). Plates were then washed and blocked with 3% BSA-PBS buffer. Serial dilutions of antibodies were added to virus-coated plates, and after washing, soluble flag-tagged NPC1 domain C protein (2 nM) was added to each well. Plates were washed and the extent of NPC1 binding to rVSV-GPCL was detected using an anti-FLAG-HRP antibody conjugate. All incubations were performed for 1 hour at 37°C. Binding was expressed as percent of the maximal binding signal obtained with no-antibody control.
Howell K.A., Qiu X., Brannan J.M., Bryan C., Davidson E., Holtsberg F.W., Wec A.Z., Shulenin S., Biggins J.E., Douglas R., Enterlein S.G., Turner H.L., Pallesen J., Murin C.D., He S., Kroeker A., Vu H., Herbert A.S., Fusco M.L., Nyakatura E.K., Lai J.R., Keck Z.Y., Foung S.K., Saphire E.O., Zeitlin L., Ward A.B., Chandran K., Doranz B.J., Kobinger G.P., Dye J.M, & Aman M.J. (2016). Antibody Treatment of Ebola and Sudan Virus Infection via a Uniquely Exposed Epitope within the Glycoprotein Receptor-Binding Site. Cell Reports, 15(7), 1514-1526.
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7

Quantifying Antibody Binding to Biotinylated VSV-GP

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Thermolysin-cleaved VSV-GP (rVSV-GPCL) was incubated with a functional-spacer-lipid reagent conjugated to biotin (FSL-biotin; Sigma) to allow incorporation into viral membrane, and the resulting biotinylated viral particles were then captured onto streptavidin-coated plates, as described previously (Ng et al., 2014 (link)). Plates were then washed and blocked with 3% BSA-PBS buffer. Serial dilutions of antibodies were added to virus-coated plates, and after washing, soluble FLAG-tagged NPC1 domain C protein (2 nM) was added to each well. Plates were washed, and the extent of NPC1 binding to rVSV-GPCL was detected using an anti-FLAG-HRP (horseradish peroxidase) antibody conjugate. All incubations were performed for 1 hr at 37°C. Binding was expressed as percentage of the maximal binding signal obtained with no-antibody control.
Howell K.A., Qiu X., Brannan J.M., Bryan C., Davidson E., Holtsberg F.W., Wec A.Z., Shulenin S., Biggins J.E., Douglas R., Enterlein S.G., Turner H.L., Pallesen J., Murin C.D., He S., Kroeker A., Vu H., Herbert A.S., Fusco M.L., Nyakatura E.K., Lai J.R., Keck Z.Y., Foung S.K., Saphire E.O., Zeitlin L., Ward A.B., Chandran K., Doranz B.J., Kobinger G.P., Dye J.M, & Aman M.J. (2016). Antibody Treatment of Ebola and Sudan Virus Infection via a Uniquely Exposed Epitope within the Glycoprotein Receptor-Binding Site. Cell Reports, 15(7), 1514-1526.
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8

Biotin-Labeling and Proteolysis of rVSV-EBOV GP Virions

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The viral envelope of rVSV-EBOV GP virions were labeled with biotin using a function-spacer-lipid construct (FSL-biotin) (Sigma-Aldrich) at neutral pH for 1 hour at 37°C, as described previously (Ng et al., 2014 ). The labeled virus stock was then acidified to pH ~5.5 by addition of 10X 2-[N-morpholino]ethanesulfonic acid (MES) buffer. Virions were diluted and aliquoted into a PCR plate. Biotin-labeled virions were pre-incubated with 1000 nM of test antibody for 1 hour at 37°C at pH 5.5. Virion-antibody complexes were then subjected to proteolysis by recombinant human Cathepsin L (R&D Systems) at 4 ng/μL for 30 minutes at 37°C. The reaction was stopped by the addition of E64 inhibitor (Peptides International) followed by dilution into 1X PBS buffer, pH 7.4. Samples were analyzed by western blot using h21D10 monoclonal antibody (Holtsberg et al., 2015 (link)) directly conjugated to horseradish peroxidase.
Zhao X., Howell K.A., He S., Brannan J.M., Wec A.Z., Davidson E., Turner H.L., Chiang C.I., Lei L., Fels J.M., Vu H., Shulenin S., Turonis A.N., Kuehne A.I., Liu G., Ta M., Wang Y., Sundling C., Xiao Y., Spence J.S., Doranz B.J., Holtsberg F.W., Ward A.B., Chandran K., Dye J.M., Qiu X., Li Y, & Aman M.J. (2017). Immunization-elicited Broadly Protective Antibody Reveals Ebolavirus Fusion Loop as a Site of Vulnerability. Cell, 169(5), 891-904.e15.
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9

Lipid Composition for Membrane Reconstitution

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1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC) and cholesterol were purchased from Avanti Polar Lipids; Texas Red 1,2dihexadecanoyl-sn-glycero-3-phosphaethanolamine (Tx-Red DHPE) from Life Technologies. The purified Gb3 extracted from red blood cells were supplied from Matreya. FSL-biotin was obtained from Sigma-Aldrich. Recombinant LecA was pro-duced from Escherichia coli according to published procedures. 32, 54 Sucrose was purchased from Carl Roth.
Omidvar R., Ayala Y.A., Brandel A., Hasenclever L., Helmstädter M., Rohrbach A., Römer W, & Madl J. (2021). Quantification of nanoscale forces in lectin-mediated bacterial attachment and uptake into giant liposomes. Nanoscale, 13(7).
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10

PUUV Glycoprotein Binding Assay

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High-protein-binding 96-well ELISA plates (Corning) were coated with purified human mAbs (40 ng per well, ‘first mAb’) at 37°C and blocked with 3% (w/v) bovine serum albumin (BSA; Thermo Fisher) in PBS. The membranes of rVSV-PUUV-Gn/Gc particles were labelled with a short-chain phospholipid probe, functional-component spacer diacyl lipid conjugated to biotin (FSL-biotin, Sigma-Aldrich), as described (30 (link)). Subsequently, pre-titrated amounts of biotin-labeled rVSVs were incubated with purified human mAb (100 nM, ‘second mAb’) for 1 h at 37°C before their addition to mAb-coated plates. Bound rVSV-PUUV-Gn/Gc was detected by incubation with a streptavidin-HRP conjugate (Thermo Scientific). Competition levels were determined by reduction of rVSV binding in presence of a competitor mAb compared to rVSV binding in the absence of competition (rVSV complexed with human IgG1 [Sigma-Aldrich]). Antibodies with >75% reduction of rVSV binding were considered to be in competition with pre-complexed mAbs.
Mittler E., Wec A.Z., Tynell J., Guardado-Calvo P., Wigren-Byström J., Polanco L.C., O’Brien C.M., Slough M.M., Abelson D.M., Serris A., Sakharkar M., Pehau-Arnaudet G., Bakken R.R., Geoghegan J.C., Jangra R.K., Keller M., Zeitlin L., Vapalahti O., Ulrich R.G., Bornholdt Z.A., Ahlm C., Rey F.A., Dye J.M., Bradfute S.B., Strandin T., Herbert A.S., Forsell M.N., Walker L.M, & Chandran K. (2022). Human antibody recognizing a quaternary epitope in the Puumala virus glycoprotein provides broad protection against orthohantaviruses. Science translational medicine, 14(636), eabl5399.
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