Nhs lc biotin

Manufactured by Thermo Fisher Scientific

Sourced in United States

NHS-LC-Biotin is a water-soluble, amine-reactive biotinylation reagent. It contains an N-hydroxysuccinimide (NHS) ester group that can form covalent bonds with primary amine groups on proteins and other biomolecules.

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Lab products found in correlation

Facscanto 2, by BD (2 mentions) Alexa fluor 568, by Thermo Fisher Scientific (2 mentions) Anti lc3 a b 4108s, by Cell Signaling Technology (1 mentions) Anti rabbit igg conjugated with horseradish peroxidase hrp, by Cell Signaling Technology (1 mentions) Celltrace violet ctv, by Thermo Fisher Scientific (1 mentions) Streptavidin peroxidase conjugate, by Roche (1 mentions) Prism 9, by GraphPad (1 mentions) Paraformaldehyde (pfa), by Merck Group (1 mentions) Streptavidin alexa fluor 647, by Thermo Fisher Scientific (1 mentions) Protein g magnetic beads, by Thermo Fisher Scientific (1 mentions) Ifn γ, by Thermo Fisher Scientific (1 mentions) Anti cd11c pe, by BD (1 mentions) 3 methyladenine, by Merck Group (1 mentions) 4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions) Propidium iodide, by Thermo Fisher Scientific (1 mentions) Extravidin r pe, by Merck Group (1 mentions) Anti human lc fitc, by Southern Biotech (1 mentions) Streptavidin agarose, by Thermo Fisher Scientific (1 mentions) Aquity uplc, by Waters Corporation (1 mentions) Lab tek 2, by Thermo Fisher Scientific (1 mentions)

17 protocols using nhs lc biotin

Immune Cell Receptor Binding Assay

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Chemicals were obtained from Sigma-Aldrich (St. Louis, MO, USA), unless otherwise stated. N-succinimidyl ester conjugated (NHS)-Alexa647 and Cell trace violet (CTV) were from ThermoFisher Scientific (Waltham, MA, USA). α-Mannan from Malassezia furfur was purchased from InvivoGen (San Diego, CA, USA). Scleroglucan (β-glucan) was obtained from Elicityl (France). Lipopolysaccharide (LPS) from Klebsiella pneumoniae O1 was obtained from Dr. Chris Whitfield (University of Guelph, Canada). Alexa647-labeled anti-mouse Dectin-1 Ab (clone 2A11, rat IgG2b) was purchased from Bio-Rad (Hercules, CA, USA). Alexa647-labeled anti-Dectin-2 Ab (clone 2B4, rat IgG2a) was generated as described previously (11 (link)). Biotinylated anti-mouse Dectin-2 Ab (clone 2B4) was also generated using NHS-LC-biotin (ThermoFisher Scientific). Alexa647-labeled isotype-control Abs, mouse Fc receptor blocking Ab (clone 93, BioLegend), and R-phycoerythrin (PE)-labeled streptavidin were purchased from BioLegend (San Diego, CA, USA). ELISA kits for mouse TNFα and IL-1β were from BioLegend and R&D systems (Minneapolis, MN, USA), respectively. Anti-LC3 A/B (#4108S), β-actin (#4970S), and anti-rabbit IgG conjugated with horseradish peroxidase (HRP) (#7074P2) were from Cell Signaling Technologies (Danvers, MA, USA).

Lamprinaki D., Beasy G., Zhekova A., Wittmann A., James S., Dicks J., Iwakura Y., Saijo S., Wang X., Chow C.W., Roberts I., Korcsmaros T., Mayer U., Wileman T, & Kawasaki N. (2017). LC3-Associated Phagocytosis Is Required for Dendritic Cell Inflammatory Cytokine Response to Gut Commensal Yeast Saccharomyces cerevisiae. Frontiers in Immunology, 8, 1397.

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SARS-CoV-2 Spike Protein Binding Assay

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Intact and glycosidase-treated
RBD, S1 subunit, and the S protein (InVivo Biotech) were biotinylated
using 10 molar excess of NHS-LC-Biotin (Thermo Scientific). Immunoassay
plates were coated with 2.5 μg/mL recombinant angiotensin-converting
enzyme 2 (ACE2, Sigma-Aldrich). Subsequently, the assay plate wells
were blocked using blocking buffer containing 1% BSA. Dilutions of
the biotinylated antigens ranging from 1 to 0.001 μg/mL were
applied to the ACE2-coated assay plate wells in duplicates. The wells
were washed three times with 250 μL of PBS containing 0.1% Tween
20. Bound biotinylated antigens were detected using a streptavidin
peroxidase conjugate (Roche) and developed with tetramethylbenzidine.
The reaction was stopped using sulfuric acid. The absorbance was measured
at 450 nm using a microplate reader. GraphPad Prism 9 was used to
plot log(dose) response curves (variable slope, four parameters) and
to compute nonlinear fits, which were utilized to calculate the half-maximal
concentrations (EC₅₀).

Gstöttner C., Zhang T., Resemann A., Ruben S., Pengelley S., Suckau D., Welsink T., Wuhrer M, & Domínguez-Vega E. (2021). Structural and Functional Characterization of SARS-CoV-2 RBD Domains Produced in Mammalian Cells. Analytical Chemistry, 93(17), 6839-6847.

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Porcine Photoreceptor Phagocytosis Assay

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Porcine eyes were obtained from abattoir and POSs were isolated and purified from porcine retinas as described previously [27 (link)]. Purified POSs were labeled with NHS-LC-Biotin (Thermo Fisher Scientific, Carlsbad, CA, USA) and resuspended in culture medium at a concentration of 5 × 10⁷ POSs/mL. Cells were incubated with POSs at 37 °C, 5% CO₂ in a humidified incubator for 4 h. Non-phagocyted or unbound POSs were removed by washing three times with PBS. Cells were fixed by 4% paraformaldehyde (PFA, Sigma) in PBS and incubated with cy3-labeled avidin (Invitrogen) for 10 min. ZO-1 immunostaining was used to show the boundary of cells. DAPI was used to label nuclei. The samples were then examined by fluorescence microscope (Olympus IX73). Z-stack images were obtained using a Nikon confocal microscope (Nikon A1R, Nikon Instruments Inc., Tokyo, Japan).

Zhu X., Chen Z., Wang L., Ou Q., Feng Z., Xiao H., Shen Q., Li Y., Jin C., Xu J.Y., Gao F., Wang J., Zhang J., Zhang J., Xu Z., Xu G.T., Lu L, & Tian H. (2022). Direct conversion of human umbilical cord mesenchymal stem cells into retinal pigment epithelial cells for treatment of retinal degeneration. Cell Death & Disease, 13(9), 785.

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PTX3 Binding to Klebsiella pneumoniae

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Binding of PTX3 to K. pneumoniae was assessed by flow cytometry and wild-field microscopy. Polyclonal rabbit anti-hPTX3 antibody (29 (link)) was biotinylated with NHS-LC-Biotin (Thermo Scientific, Waltham, USA), according to the manufacturer’s instructions. 10⁶ CFSE-labelled K. pneumoniae were incubated with PTX3 (ranging from 1 to 20 μg/ml) in PBS²⁺ containing 0.2% (w/v) BSA for 1h at room temperature. Unbound PTX3 was removed by washing. The samples were then incubated with anti-hPTX3 biotinylated polyclonal antibody (0.5 μg/ml) followed by incubation with Streptavidin-Alexa Fluor 647 (Invitrogen™) (1:500) to detect bound proteins. Samples were fixed with 1% PFA and analyzed either on a FACS Canto II flow cytometry system (BD Biosciences, East Rutherford, NJ) or wild-field microscopy. FITC-labeled Aspergillus fumigatus conidia were used as positive control (28 (link)).

Asgari F., Supino D., Parente R., Polentarutti N., Stravalaci M., Porte R., Pasqualini F., Barbagallo M., Perucchini C., Recordati C., Magrini E., Mariancini A., Riva F., Giordano A., Davoudian S., Roger T., Veer C.V., Jaillon S., Mantovani A., Doni A, & Garlanda C. (2021). The Long Pentraxin PTX3 Controls Klebsiella Pneumoniae Severe Infection. Frontiers in Immunology, 12, 666198.

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Antibody Conjugation with Biotin and Fluorophores

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Antibodies were labeled with biotin or organic fluorophores via N-hydroxysuccinimide (NHS)–reactive ester. For biotin conjugation, antibodies (0.1 to 1 mg/ml) were incubated with 50-fold molar excess of succinimidyl-6-(biotinamido)hexanoate (NHS-LC-biotin) (Thermo Fisher Scientific) for 30 to 60 min and then isolated on 7-kDa gel filtration columns (Thermo Fisher Scientific). For dye conjugation, antibodies were precaptured on protein G magnetic beads (Thermo Fisher Scientific) and incubated with 10-fold molar excess of Alexa Fluor dye–NHS (Thermo Fisher Scientific) for 30 to 60 min. Free dye was washed out, and antibodies were further purified on 7-kDa gel filtration columns. The degree of labeling and the concentration of antibodies were measured by spectrophotometry.

Mao C.P., Wang S.C., Su Y.P., Tseng S.H., He L., Wu A.A., Roden R.B., Xiao J, & Hung C.F. (2021). Protein detection in blood with single-molecule imaging. Science Advances, 7(33), eabg6522.

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Purification and Biotinylation of CRP

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Human CRP and recombinant CRP were purified as described previously (18 (link)). Rat CRP was purified using phosphorylcholine (PCh) Sepharose by the same protocol. CRP was biotinylated at a neutral pH as described for the reagent NHS-LC-biotin (Thermofisher) to prevent CRP denaturation. The CRP biotin was repurified on PCh-Sepharose to remove non-binding protein with extensive washing to remove unreacted biotin.

Seow E.S., Doran E.C., Schroeder J.H., Rogers M.E, & Raynes J.G. (2023). C-reactive protein binds to short phosphoglycan repeats of Leishmania secreted proteophosphoglycans and activates complement. Frontiers in Immunology, 14, 1256205.

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Biotinylation of Monoclonal Antibodies

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Monoclonal antibody Y-Ae was purified from hybridoma culture supernatants by standard protein G column chromatography (Amersham Biosciences/GE Healthcare). Purified mAbs (2.0 mg ml⁻¹ in PBS) were biotinylated overnight at 4 °C using NHS–LC–biotin (N-Hexanoate-succinimidyl-long chain-biotin, Thermo scientific), dissolved to 10 mM with DMSO just before use, with a biotin-to-mAb molar ratio of 30:1. Unbound biotin was not removed, as this did not interfere with downstream applications. Anti-I-A/I-E-FITC (fluorescein isothiocyanate ) and anti-CD11c-PE mAbs were obtained from BD Biosciences. Rapamycin was from Calbiochem and IFNγ was from Peprotech. 3-Methyladenine was from Sigma. The nuclear fluorescent dye 4,6-diamidino-2-phenylindole (DAPI) was purchased from Sigma. Anti-LC3 antibodies for western blotting and immunofluorescence analysis were purchased from Novus Biologicals and Cell Signaling, respectively. Antibodies to LAMP-1, β-actin and p62 were purchased from Abcam.

Saini N.K., Baena A., Ng T.W., Venkataswamy M.M., Kennedy S.C., Kunnath-Velayudhan S., Carreño L.J., Xu J., Chan J., Larsen M.H., Jacobs WR J.r, & Porcelli S.A. (2016). Suppression of autophagy and antigen presentation by Mycobacterium tuberculosis PE_PGRS47. Nature microbiology, 1(9), 16133.

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Porcine Retinal Pigment Epithelial Phagocytosis

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Porcine eyes were obtained from abattoir and POSs were isolated and purified from porcine retinas as described previously (Hu et al., 2020 (link)). Purified POSs were labeled with NHS-LC-Biotin (Thermo Fisher Scientific, Newton Drive, Carlsbad) and resuspended in culture medium at a concentration of 5×10⁷ POSs/mL. RPE cells were incubated with POSs at 37°C, 5% CO₂ in a humidified incubator for 4h. Non-phagocyted POSs were removed by washing three times with PBS. Cells were fixed by 4% paraformaldehyde (PFA, Sigma) in PBS and incubated with FITC-labeled or CY3-labeled avidin (Invitrogen) for 10min. ZO-1 immunostaining was used to show the boundary of cells. DAPI was used to label nuclei. The samples were then examined by fluorescence microscope (Olympus IX73).

Tian H., Chen Z., Zhu X., Ou Q., Wang Z., Wu B., Xu J.Y., Jin C., Gao F., Wang J., Zhang J., Zhang J., Lu L, & Xu G.T. (2022). Induced retinal pigment epithelial cells with anti-epithelial-to-mesenchymal transition ability delay retinal degeneration. iScience, 25(10), 105050.

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Polyspecificity Antibody Binding Assay

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Polyspecificity reagent binding of antibodies was performed as described previously (37 (link)). Briefly, soluble membrane protein (SMP) and soluble cytosolic protein (SCP) fractions were extracted from Chinese hamster ovary (CHO) cells and biotinylated using NHS-LC-Biotin (Thermo Fisher Scientific) reagent. Yeast-presented IgGs were incubated with 1:10 diluted stock of biotinylated SMP and SCP for 20 minutes on ice, followed by two washes with PBSF, and stained with 50 μL of a secondary labeling mix containing ExtrAvidin-R-PE (Sigma-Aldrich), anti-human LC-FITC (Southern Biotech), and propidium iodide (Invitrogen) for 15 minutes on ice. Cells were subsequently washed with PBSF and resuspended in PBSF for flow cytometric analysis on a BD FACS Canto II (BD Biosciences). Polyreactivity scores were also reported for 42 previously described clinical antibodies for comparison (38 (link)).

Rappazzo C.G., Tse L.V., Kaku C.I., Wrapp D., Sakharkar M., Huang D., Deveau L.M., Yockachonis T.J., Herbert A.S., Battles M.B., O’Brien C.M., Brown M.E., Geoghegan J.C., Belk J., Peng L., Yang L., Scobey T.D., Burton D.R., Nemazee D., Dye J.M., Voss J.E., Gunn B.M., McLellan J.S., Baric R.S., Gralinski L.E, & Walker L.M. (2020). An Engineered Antibody with Broad Protective Efficacy in Murine Models of SARS and COVID-19. bioRxiv.

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Biotin-Streptavidin Coupling Reactions

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Coupling reactions were performed under an Ar atmosphere using dry solvents. All commercially available reagents were purchased from Sigma-Aldrich and were used as received. NHS-LC-biotin and streptavidin agarose beads were purchased from Thermo Scientific. Spin ultrafilters (vivaspin 500) were purchased from GE Healthcare. ¹H and ¹³C NMR spectra were recorded on Bruker instruments (400 or 500 MHz for ¹H and 100 or 125 MHz for ¹³C). MS data were collected with AQUITY UPLC from Waters.

Lee S., Wang W., Lee Y, & Sampson N.S. (2015). Cyclic acetals as cleavable linkers for affinity capture. Organic & biomolecular chemistry, 13(31), 8445-8452.

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