Biotin n hydroxysuccinimide ester

Manufactured by Merck Group

Sourced in United States

Biotin N-hydroxysuccinimide ester is a chemical compound used in various biotechnological and biochemical applications. It is a reactive form of biotin that can be easily conjugated to other molecules, such as proteins, peptides, or nucleic acids. The N-hydroxysuccinimide (NHS) ester group enables the biotin to form stable amide bonds with amine-containing substances.

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Close spelling variants of this product

Biotin 3-sulfo-N-hydroxysuccinimide ester sodium salt (4 citations) (+)-biotin N-hydroxysuccinimide ester (biotin-NHS; (4 citations) N-hydroxysuccinimide (757 citations)

24 protocols using biotin n hydroxysuccinimide ester

Sandwich ELISA for Antibody Detection

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Biotin N-hydroxysuccinimide ester and streptavidin were from Sigma-Aldrich. Goat anti-mouse IgG and IgM polyclonal antibodies were from Jackson ImmunoResearch. Rat anti-mouse IgG1, IgG2a and IgG2b antibodies were from AbD Serotec. Goat anti-mouse IgA antibodies were from CliniSciences. Sandwich ELISAs were performed with MaxiSorp 96-well microtiter plates (Nunc, Thermoscientific), and all reagents were diluted in Enzyme ImmunoAssay (EIA) buffer (0.1 M phosphate buffer [pH 7.4] containing 0.15 M NaCl, 0.1% bovine serum albumin [BSA], and 0.01% sodium azide). AEBSF (serine protease inhibitor) was from Interchim. Dialysis membranes were from Spectra/Por. Cholera Toxin and Luria Broth were from Sigma. PBS was from Gibco by Life Technologies.

Jneid B., Moreau K., Plaisance M., Rouaix A., Dano J, & Simon S. (2016). Role of T3SS-1 SipD Protein in Protecting Mice against Non-typhoidal Salmonella Typhimurium. PLoS Neglected Tropical Diseases, 10(12), e0005207.

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HTLV-1 Impact on RA T-Cell Phenotypes

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For HAS-Flow analysis, whole blood samples in EDTA tubes were obtained periodically from 57 participants with HTLV-1-positive RA and 13 participants with HTLV-1-negative RA. HAS-Flow analysis was performed to detect the expression levels of CADM1 and CD7 in CD4+ T-lymphocytes, according to previously reported methods [11 (link)]. An unlabelled CADM1 antibody (clone 3E1) was purchased from MBL (Tokyo, Japan) and subjected to primary amine biotinylation using biotin N-hydroxysuccinimide ester (Sigma Aldrich, St. Louis, MO, USA). All other antibodies were obtained from BioLegend (San Diego, CA, USA). Cells were stained using a combination of biotin-CADM1, allophycocyanin-CD7, and PE-Cy7-CD4. After washing, phycoerythrin (PE)-conjugated streptavidin was applied. A FACS Calibur instrument (BD Immunocytometry Systems, San Jose, CA, USA) was used for all multicolour flow cytometry. Data were analyzed using the FlowJo software (TreeStar, San Carlos, CA, USA).

Umekita K., Hashikura Y., Takaki A., Kimura M., Kawano K., Iwao C., Miyauchi S., Kawaguchi T., Matsuda M., Hashiba Y, & Hidaka T. (2023). HAS-Flow May Be an Adequate Method for Evaluating Human T-Cell Leukemia Virus Type 1 Infected Cells in Human T-Cell Leukemia Virus Type 1-Positive Rheumatoid Arthritis Patients Receiving Antirheumatic Therapies: A Retrospective Cross-Sectional Observation Study. Viruses, 15(2), 468.

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Recombinant Protein Expression and Purification

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Oligonucleotides were custom-synthesized by Sigma-Aldrich (Bengaluru, India). Deep-vent DNA polymerase, plasmid pMAL-c4X, Bam H1 and Eco R1 restriction enzymes, T4 DNA ligase, amylose resin, factor Xa, and EK12 cells were purchased from New England Biolabs (Hitchin, Hertfordshire, UK). Isopropyl thiogalactoside (IPTG), avidin-peroxidase, 3,3'-diaminobenzidine, gelatin, tricine, triton X-100, biotin N-hydroxysuccinimide ester, protein A-Sepharose, polyvinylidine difluoride (PVDF) membrane, Freund's Complete and Incomplete Adjuvants were procured from Sigma Aldrich (Bengaluru). Centrifugal concentrator units were purchased from Sartorius (Gottingen, Germany). All other reagents used were of analytical grade and obtained locally. CSF samples were collected into sterile tubes and placed on ice, and within 1 h of collection, frozen and preserved at −80°C until analyses were obtained from the archives of the Human Brain Tissue Repository (HBTR), NIMHANS. This study was approved by the Institutional Human Ethics Committee.

Subramanian S., Mahadevan A., Satishchandra P, & Shankar S.K. (2016). Development of a dot blot assay with antibodies to recombinant “core” 14-3-3 protein: Evaluation of its usefulness in diagnosis of Creutzfeldt–Jakob disease. Annals of Indian Academy of Neurology, 19(2), 205-210.

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Targeted Delivery of Quantum Dots

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All purchased chemicals were used without further purification. Streptavidin-modified QDs emitting at 655 nm (QDSV) were purchased from Invitrogen (Carlsbad, CA). Biotin-N-hydroxysuccinimide ester (98% HPLC purified) and N-(3-dimethylaminopropyl)-N^’-ethylcarbodiimide hydrochloride (EDC), ethylenediamine core generation-5 polyamidoamine dendrimer (G5-PAMAM), and fetal bovine serum (FBS) were purchased from Sigma-Aldrich. Targeted peptide (AKXVAAWTLKAAAZC)-G5 dendrimer conjugate was purchased from 21^st Century Biochemicals. 96-well microtiter plates with non-binding surface were purchased from Corning (NY). Microcon YM-100 (100 kDa MWCO) spin-columns were purchased from Millipore (Billercia, MA). Native coelenterazine was obtained from Prolume (Pinetop, AZ). Zeba desalting spin columns (7 kDa MWCO), monobasic sodium phosphate anhydrous, and dibasic sodium phosphate heptahydrate were purchased from Thermo Fischer Scientific (Rockford, IL). Dulbecco’s modified Eagle’s medium/Ham’s F12 50/50 mix, 1% antibiotic-antimycotic solution, and trypsin-EDTA were purchased from Cellgro (Manassas, VA).

Kumar M., Kovalski L., Broyles D., Hunt E.A., Daftarian P., Diciki E., Daunert S, & Deo S.K. (2016). Design and Development of High Bioluminescent Resonance Energy Transfer Efficiency Hybrid-Imaging Constructs. Analytical biochemistry, 498, 1-7.

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Quantifying EGFR Binding of Therapeutic mAbs

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To assess the binding of cetuximab and panitumumab to EGFR expressing target cells, anti-EGFR mAbs were concentrated using Amicon ultra centrifuge 0.5ml 30K tubes (EMD Millipore, Netherlands). Concentrations of the mAbs were adjusted to 20mg/ml and then biotinylated using Biotin-N-hydroxysuccinimide ester (Sigma Aldrich, St Louis, USA) according to the manufacturer’s instructions. The biotinylated anti-EGFR mAbs were incubated with A431 (1x10⁶) cells for 1hr, washed twice in ice cold PBS and stained with streptavidin APC (BD biosciences, Netherlands). A nonspecific IgG₁ and IgG₂ specific APC labeled antibody was used as a negative control.

Veluchamy J.P., Spanholtz J., Tordoir M., Thijssen V.L., Heideman D.A., Verheul H.M., de Gruijl T.D, & van der Vliet H.J. (2016). Combination of NK Cells and Cetuximab to Enhance Anti-Tumor Responses in RAS Mutant Metastatic Colorectal Cancer. PLoS ONE, 11(6), e0157830.

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Identifying CADM1+ Immune Cells

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Peripheral blood mononuclear cells were isolated from whole blood by density gradient centrifugation, as described previously.8 (link) An unlabeled CADM1 antibody (clone 3E1) and an isotype control chicken IgY antibody were purchased from MBL (Nagoya, Japan). These were biotinylated (by primary amine biotinylation) using biotin N-hydroxysuccinimide ester (Sigma Aldrich, St. Louis, MO, USA). A Pacific Orange-conjugated anti-CD14 antibody was purchased from Life Technologies (Carlsbad, CA, USA). All other antibodies were obtained from BioLegend (San Diego, CA, USA). Cells were stained using a combination of biotin-CADM1, allophycocyanin (APC)-CD7, APC-Cy7-CD3, Pacific Blue-CD4 and Pacific Orange-CD14. After washing, phycoerythrin (PE)-conjugated streptavidin was applied. Propidium iodide (PI [Sigma Aldrich]) was added to the samples to stain dead cells immediately before flow cytometry. A FACSAria instrument (BD Immunocytometry Systems, San Jose, CA, USA) was used for all multicolor flow cytometry and fluorescence-activated cell sorting (FACS). Data were analyzed using the FlowJo software (TreeStar, San Carlos, CA, USA). The gating procedure was as described previously.10 (link) Briefly, PI⁺ cells and then CD14⁺ cells were gated out. Next, a CADM1 versus CD7 plot for CD4⁺ cells was constructed (Fig.1a).

Kobayashi S., Watanabe E., Ishigaki T., Ohno N., Yuji K., Nakano K., Yamochi T., Watanabe N., Tojo A., Watanabe T, & Uchimaru K. (2015). Advanced human T-cell leukemia virus type 1 carriers and early-stage indolent adult T-cell leukemia-lymphoma are indistinguishable based on CADM1 positivity in flow cytometry. Cancer Science, 106(5), 598-603.

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Protein-Ligand Binding Kinetics by BLI

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Experiments were performed using the Octet RED96 Bio-Layer Interferometry. The target of interest is biotinylated either through sortagging or labeled with (+)-Biotin N-hydroxysuccinimide ester (Sigma-Aldrich). For NHS labeling, 2-fold molar excess of biotin-NHS was used for labeling at room temperature for 1 hour in PBS. Excess biotin-NHS was removed using ZeBa columns (7K MWCO, Thermo Fisher). All octet measurements were taken in 1% BSA in PBS with 1% Tween. 10 ng/mL of biotin-labeled target were loaded on Dip and Read™ Streptavidin (SA) Biosensors (Pall Fortebio) until the reading exceeded 1 nm. These sensors were then dipped into wells containing different concentrations of VHH05, ranging from 50 nM to 8 μM. Average and standard deviations were taken for at least 3 measurements.

Ling J., Cheloha R.W., McCaul N., Sun Z.Y., Wagner G, & Ploegh H.L. (2019). A nanobody that recognizes a 14-residue peptide epitope in the E2 ubiquitin-conjugating enzyme UBC6e modulates its activity.. Molecular immunology, 114, 513-523.

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Site-Specific Labeling of Lipoprotein Lipase

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Single cysteine mutants of LPL were purified (final buffer composition was 1.5 M NaCl, 10% glycerol, 20 mM Bis-Tris pH 6.5) and incubated with an approximately 10-fold excess of Alexa Fluor® Maleimide Dyes (Invitrogen). Label was added dropwise to the protein solution followed by gentle mixing. The reaction was allowed to continue, in light protected tubes, for 45 minutes at 4 °C or on ice. For samples in which biotinylated LPL was used, a 1–2 molar excess of Biotin-N-Hydroxysuccinimide ester (Sigma) was next added and the reaction was allowed to proceed on ice for another 10 minutes. For both biotinylated and non-biotinylated samples, LPL was then diluted to less than 500 mM NaCl with 20 mM Bis-Tris pH 6.5 and re-purified over a HiTrap Heparin Sepharose High Performance Column (GE Healthcare Life Sciences) to remove excess biotin and/or label. Excess label flowed through the heparin column whereas purified, labeled LPL bound and eluted normally. Alternatively, labeling could be followed by removal of excess dye by use of two tandem 7 kDa Zebra desalting columns (ThermoFisher Scientific). LPL samples were aliquoted and stored at −80 °C until use. If samples were concentrated, the samples were syringe filtered through a 0.2 μm PDVF filter to remove aggregates.

Hayne C.K., Yumerefendi H., Cao L., Gauer J.W., Lafferty M.J., Kuhlman B., Erie D.A, & Neher S.B. (2018). We FRET So You Don’t Have To: New Models of the Lipoprotein Lipase Dimer. Biochemistry, 57(2), 241-254.

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Fluorescent Labeling of Anti-Collagen-1 Antibody

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Anti-collagen-1 rabbit pAb (AbCam, Cambridge, UK) was prelabeled with a fluorescent tag and a biotin group to ensure both its fluorescent visibility in nanopatterns and cross-linking to the streptavidin-functionalized PAA gels, respectively. Briefly, 20μL of 1mg/mL antibody sample was incubated for 1 hour with 5μL of ((+)-biotin N-hydroxysuccinimide ester, Sigma-Aldrich; as per the commercial protocol) and 5μL of fluorescent tag kit (Alexa Fluor^® succinimidyl esters, Invitrogen, Molecular Probes ; as per the commercial protocol). Labeled protein then was dialysed overnight in Slide-A-Lyzer MINI Dialysis Device, 7K MWCO (Thermo Fisher) overnight at 4° C in cold PBS, then stored at 4° C in the darkness. 10μL droplets of 0.1mg/mL labeled antibody solution were then placed atop of the 5×5mm or 1×1cm square micro- or nano-stamps. To ensure a proper coverage and effective stamp surface coating with labeled α-collagen-1 antibody, the antibody solution droplet was ‘‘sandwiched’’ between the stamp’s printing surface and 15mm round glass coverslip (Carolina, USA), which had been baked in the furnace for 5–10 hours at 450° C.

Tabdanov E.D., Puram V., Zhovmer A, & Provenzano P.P. (2018). Microtubule-Actomyosin Mechanical Cooperation during Contact Guidance Sensing. Cell reports, 25(2), 328-338.e5.

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Isolation and Analysis of Pancreatic Immune Cells

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Bone marrow was eluted from long bones, and spleens and draining pancreatic lymph nodes were macerated with HBSS (Invitrogen) plus 10% FBS (HyClone). RBCs were lysed using Tris-NH₄Cl. Freshly isolated pancreata were digested with 3 ml 1 mg/ml collagenase P diluted in HBSS for 30 min at 37°C, and tissue was disrupted using an 18-gauge needle. Ice-cold HBSS plus 10% FBS was added immediately to inhibit collagenase activity. Cells were directly analyzed by flow cytometry. Flow cytometry Ab reagents were reactive with B220 (6B2), IgM^a (DS-1), CD19 (1D3), CD21 (7G6), CD23 (B3B4), CD93 (AA4.1) (BD Biosciences), or IgM (μ-chain specific) (Life Technologies). Biotin N-hydroxysuccinimide ester was used to biotinylate human insulin (both from Sigma) at pH 8 in bicine buffer. Streptavidin reagents (BD Biosciences) were used to detect biotinylated reagents. 7-Aminoactinomycin D (BD Biosciences) was used to exclude dead cells. Sample acquisition was performed using an LSR II flow cytometer (BD Biosciences), and FlowJo software (TreeStar) was used for analysis.

Bonami R.H., Sullivan A.M., Case J.B., Steinberg H.E., Hoek K.L., Khan W.N, & Kendall P.L. (2014). Bruton’s Tyrosine Kinase Promotes Persistence of Mature Anti-Insulin B Cells. Journal of immunology (Baltimore, Md. : 1950), 192(4), 1459-1470.

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