Hrp conjugated streptavidin

Manufactured by Merck Group

Sourced in United States

HRP-conjugated streptavidin is a protein complex consisting of streptavidin, a tetrameric protein that binds strongly to the vitamin biotin, conjugated with horseradish peroxidase (HRP), an enzyme commonly used in various immunoassays and detection methods. This product can be used for the detection and quantification of biotinylated molecules in various applications.

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Lab products found in correlation

3 3 diaminobenzidine (dab), by Merck Group (3 mentions) Ez link nhs biotin, by Thermo Fisher Scientific (2 mentions) O phenylenediamine dihydrochloride, by Merck Group (2 mentions) Spectramax m2, by Molecular Devices (2 mentions) Entellan, by Merck Group (2 mentions) Maxisorp plate, by Thermo Fisher Scientific (2 mentions) High protein binding plates, by Corning (2 mentions) Microplate reader, by Thermo Fisher Scientific (2 mentions) O phenylenediamine substrate, by Merck Group (1 mentions) Hrp opteia, by BD (1 mentions) Biotin conjugated goat anti mouse igg, by Merck Group (1 mentions) Anti type 1 collagen, by Merck Group (1 mentions) Anti collagen type 3, by Abcam (1 mentions) Bvd6 24g2, by BioLegend (1 mentions) Il 12p40, by BD (1 mentions) Mouse ril 4, by Thermo Fisher Scientific (1 mentions) Mouse ifn γ, by R&D Systems (1 mentions) Tween 20, by Merck Group (1 mentions) Tmb substrate, by BD (1 mentions) H2so4, by Merck Group (1 mentions)

38 protocols using hrp conjugated streptavidin

Pericardial Tissue Histology and Glycan Analysis

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Fresh and glutaraldehyde fixed pericardial samples were embedded in Optimal Cutting Temperature material and cryosectioned (8 μm). Sections were air dried, fixed with acetone for 10 min and stored at −80 °C until used. Fresh and glutaraldehyde fixed samples were also fixed in formalin and paraffin embed for Masson’s Trichrome histology staining. Tissue sections were stained to detect the Gal antigen and collagens. Fresh and glutaraldehyde fixed tissues were stained with biotin-conjugated GSIB-4 (3 μg/ml) and murine monoclonal antibodies (2 μg/ml) to Collagen I (Anti-Collagen Type I, Sigma-Aldrich), III (Anti-Collagen Type III, Abcam, Cambridge, UK), and V (Anti-Collagen Type V, Abcam). Lectin and antibody solutions were produced in dPBS with 1% BSA and primary incubations were at 4 °C for 12 h. GSIB-4 binding was detected with HRP-conjugated streptavidin (Sigma-Aldrich) and mouse anti-collagen antibody binding was detected with biotin conjugated goat anti-mouse IgG (Sigma-Aldrich) and HRP-conjugated streptavidin. All sections were incubated with 3,3′-Diaminobenzidine (Sigma-Aldrich) and counter stained with haematoxylin.

McGregor C., Byrne G., Rahmani B., Chisari E., Kyriakopoulou K, & Burriesci G. (2016). Physical equivalency of wild type and galactose α 1,3 galactose free porcine pericardium; a new source material for bioprosthetic heart valves. Acta Biomaterialia, 41, 204-209.

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Protein Binding Quantification Assay

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Purified CH2 domain proteins in the concentration of 2 mg/ml were coated on 96-well ELISA plates overnight at 4 ° C. Serially concentrations of biotinylated proteins with triplicate samples were added into wells and incubated for 1 h. Bound proteins were detected with HRP conjugated streptavidin (1:1000; Sigma). The o- Phenylenediamine substrate (Sigma) was added and the reaction was read at 450 nm.

Li D., Gong R., Zheng J., Chen X., Dimitrov D.S, & Zhao Q. (2017). Engineered antibody CH2 domains binding to nucleolin: Isolation, characterization and improvement of aggregation. Biochemical and biophysical research communications, 485(2), 446-453.

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Cytokine Profiling of CAR-T Cell Culture

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Approximately 1 × 10⁶ CAR‐T cells were cultured in 1 mL of medium (without IL‐2 supplementation) in 12‐well flat‐bottom plates for 48 h. Triplicate wells were plated for each condition. The supernatants were collected and analyzed using a human ELISA kit (MLUTI SCIENCES, China). ELISAs were performed using purified anti‐IL‐2, anti‐IFN‐γ, anti‐TNF‐α, and anti‐Granzyme B mAbs as capture Abs; the corresponding biotinylated anti‐IL‐2, anti‐IFN‐γ, anti‐TNF‐α, and anti‐Granzyme B mAbs; HRP‐conjugated streptavidin (Sigma Aldrich); and tetramethylbenzidine microwell peroxidase substrate and stop solution (Kirkegaard and Perry Laboratories) according to the manufacturer's instructions.

Shao M., Teng X., Guo X., Zhang H., Huang Y., Cui J., Si X., Ding L., Wang X., Li X., Shi J., Zhang M., Kong D., Gu T., Hu Y., Qian P, & Huang H. (2022). Inhibition of Calcium Signaling Prevents Exhaustion and Enhances Anti‐Leukemia Efficacy of CAR‐T Cells via SOCE‐Calcineurin‐NFAT and Glycolysis Pathways. Advanced Science, 9(9), 2103508.

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Antibody Binding Affinity Determination

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The EC₅₀ was determined using ELISA on B-type RBCs, which were immobilized on the ELISA plates as described by Bigbee et al.63 (link). Endogenous RBC peroxidase was blocked with 3% H₂O₂ for 20 min. The immobilized RBCs were incubated for 2 h with two-fold serial dilutions of HEK293 culture media containing transiently expressed M18_IgG3 and O10_IgG3 antibodies. The antibody concentrations in the media were determined using an IgG3-specific ELISA. The amounts of antibodies bound to RBCs were detected with biotinylated goat anti-mouse kappa polyclonal antibodies (1:3000, AbD Serotec), HRP-conjugated streptavidin (1:40000, Sigma) and a colorimetric substrate for HRP OptEIA (BD Bioscience). The obtained absorbance values were plotted against concentrations of the antibodies using an on-line tool ( www.ic50.tk), which was also used for the EC₅₀ calculations.

Klaus T., Bzowska M., Kulesza M., Kabat A.M., Jemioła-Rzemińska M., Czaplicki D., Makuch K., Jucha J., Karabasz A, & Bereta J. (2016). Agglutinating mouse IgG3 compares favourably with IgMs in typing of the blood group B antigen: Functionality and stability studies. Scientific Reports, 6, 30938.

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ELISA-Based Cytokine Quantification in Mouse Serum

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Mouse IFN-γ (R&D Systems) and IL-12p40 (BD Biosciences) ELISA kits were used according to the manufacturer’s protocols to determine cytokine concentrations in mouse serum. Serum concentrations of IL-4 were determined using a sandwich ELISA. Maxisorp plates (Nunc) were coated with anti-mouse IL-4 capture antibody (2 µg/mL; 11B11; BioLegend) in 50 mM sodium bicarbonate buffer (pH 9.4) overnight. Plates were washed with 0.05% Tween 20 (Sigma-Aldrich) in PBS, blocked with 4% bovine serum albumin (BSA; Roche) in PBS for 2 h, and then samples and standards (mouse rIL-4; Peprotech), serially diluted two-fold from 2,000 pg/mL, were added for 1 h. Anti-mouse IL-4 detection antibody (0.5 µg/mL; BVD6-24G2, BioLegend) diluted in 4% BSA in PBS was added for 1 h, followed by HRP-conjugated streptavidin (2 µg/mL; Sigma-Aldrich) for 30 min. The plates were developed with TMB substrate (BD Biosciences), and the reaction stopped with 1 M H₂SO₄ (Sigma-Aldrich).

Kling J.C., Jordan M.A., Pitt L.A., Meiners J., Thanh-Tran T., Tran L.S., Nguyen T.T., Mittal D., Villani R., Steptoe R.J., Khosrotehrani K., Berzins S.P., Baxter A.G., Godfrey D.I, & Blumenthal A. (2018). Temporal Regulation of Natural Killer T Cell Interferon Gamma Responses by β-Catenin-Dependent and -Independent Wnt Signaling. Frontiers in Immunology, 9, 483.

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Glycoconjugate Profiling by Periodate-Schiff Staining

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Wild-type epimastigote forms (1–3 x 10⁹) were delipidated by chloroform/methanol/water (5:10:4 v/v/v) treatment and then subjected to successive butan-1-ol/water partitions as described [43 (link)]. Aliquots of fractions enriched in glycoconjugates were resolved by SDS-PAGE, transferred to nitrocellulose membranes (GE Healthcare), and subjected to a slightly modified version of the periodate-Schiff staining technique. Briefly, blots were incubated in the dark for 1 h in 0.1 M acetic acid containing 10 mM sodium periodate. After extensive washings with PBS, membranes were incubated for 5 min in 15 mM glycerol solution followed by 2 h-incubation in the dark with 5 mM biotin hidrazide (Sigma). Membranes were extensively washed with PBS, blocked with PBS supplemented with 0.1% (v/v) Tween 20 and 5% (w/v) Bovine Serum Albumin (BSA), incubated with HRP-conjugated streptavidin (1:5,000; Sigma) for 1 h and developed using Super Signal West Pico chemiluminescent substrate (Thermo Scientific).

Cámara M.D., Balouz V., Centeno Cameán C., Cori C.R., Kashiwagi G.A., Gil S.A., Macchiaverna N.P., Cardinal M.V., Guaimas F., Lobo M.M., de Lederkremer R.M., Gallo-Rodriguez C, & Buscaglia C.A. (2019). Trypanosoma cruzi surface mucins are involved in the attachment to the Triatoma infestans rectal ampoule. PLoS Neglected Tropical Diseases, 13(5), e0007418.

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Enzymatic Reaction of OPD with HRP

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OPD dihydrochloride is often used as a substrate in enzymatic reactions with horseradish peroxidase in ELISA.^[²⁸^] A 5 mg OPD tablet (Sigma‐Aldrich) was dissolved in 12.5 mL citric acid‐phosphate buffer at pH 5 (0.4 mg OPD mL⁻¹; OPD solution). HRP‐conjugated streptavidin (Sigma‐Aldrich) was dissolved in phosphate‐buffered saline at pH 7.4 (PBS) (0.01 mg mL⁻¹; HRP solution). OPD solution (23 µL) was mixed with HRP solution (0.5 µL). A portion (10 µL) of the mixed solution was levitated, and 0.8 µL of 0.3% H₂O₂ was added (0 min). After 15 min, the solution changed color to orange‐brown, and a 44% decrease in its volume was observed (6.0 µL final volume).

Matsubara T, & Takemura K. (2020). Containerless Bioorganic Reactions in a Floating Droplet by Levitation Technique Using an Ultrasonic Wave. Advanced Science, 8(3), 2002780.

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Biotinylation of Protein Antigens

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Proteins 350 and 780 were constructed and expressed as previously reported (Gray et al. 2012 (link)). Proteins 030 and 14-3-3 were obtained from the Seattle Structural Genomics Center for Infectious Disease (SSGCID). All four antigens were biotinylated using either the Pierce EZ-Link NHS-Peg 4–Biotin or the Sulfo-NHS-LC-Biotin kit (Thermo Scientific, Rockford, IL). Biotinylation was confirmed by Western blots probed with 0.4 ug/mL HRP-conjugated streptavidin (Sigma Aldrich, St. Louis, MO).

Kahn M., Priddy S., Estrada M., Spadafora L., Cangelosi G.A, & Domingo G.J. (2015). Methodology for preservation of yeast-bound single chain fragment variable antibody affinity reagents. Journal of immunological methods, 427, 134-137.

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Cross-competition ELISA for bNAb Binding

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D11A.B5, D11A.F2, and D11A.F9 were biotinylated using EZ-Link NHS-Biotin (Pierce Biotechnology, Thermo Scientific) per the manufacturer s protocol. For cross-competition ELISA assays, 16055 NFL TD CC trimers were captured on the ELISA plate by a mouse anti-His tag mAb (R&D Systems) coated at 2 μg/ml in PBS at 4°C overnight. Five-fold serial dilutions of various bNAbs and non-bNAbs were pre-incubated with the captured trimer at RT for 30 min prior to addition of the biotinylated mAbs at a concentration previously determined to give ~75% of the maximum binding signal (i.e. binding to trimer with no competitor present) for 60 min at RT. The bound biotinylated mAbs were detected using HRP-conjugated streptavidin (Sigma). The signal was detected with TMB substrate and reaction stopped with 1 N sulfuric acid as above. Wells were blocked with 5% non-fat milk in PBS-T (0.2% Tween-20) after coating with the anti-His mAb and washed with PBS-T after each incubation step. Samples were diluted in 10% blocking buffer. Competition is expressed as percent inhibition where 0% was the absorbance measured with no inhibitor present.

Martinez-Murillo P., Tran K., Guenaga J., Lindgren G., Àdori M., Feng Y., Phad G.E., Bernat N.V., Bale S., Ingale J., Dubrovskaya V., O’Dell S., Pramanik L., Spångberg M., Corcoran M., Loré K., Mascola J.R., Wyatt R.T, & Karlsson Hedestam G.B. (2017). Particulate array of well-ordered HIV clade C Env trimers elicits neutralizing antibodies that display a unique V2 cap approach. Immunity, 46(5), 804-817.e7.

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Biotin-Conjugated GNA Detection

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Alternatively, SDS-PAGE resolved proteins were transferred to Immobilon-P membranes. The membranes were blocked with TBS containing 3% BSA for 1 h and then incubated for 2 h with 1 µg/mL of biotin-conjugated GNA (EY Laboratories, San Mateo, CA, USA) in T-TBS. Membranes were washed three times for 15 min each with T-TBS and incubated for 1 h with 0.5 µg/mL of HRP-conjugated streptavidin (Sigma-Aldrich) in T-TBS. Membranes were then washed three times for 15 min each with T-TBS, followed by color development using TMB.

Dilimulati K., Orita M., Yonahara Y., Imai F.L, & Yonezawa N. (2022). Identification of Sperm-Binding Sites in the N-Terminal Domain of Bovine Egg Coat Glycoprotein ZP4. International Journal of Molecular Sciences, 23(2), 762.

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