Streptavidin hrp conjugate

Manufactured by Merck Group

Sourced in United States

Streptavidin-HRP conjugate is a laboratory reagent that is composed of the protein streptavidin covalently linked to the enzyme horseradish peroxidase (HRP). Streptavidin, a protein derived from the bacterium Streptomyces avidinii, has a high affinity for the small molecule biotin. The HRP enzyme is capable of catalyzing a colorimetric reaction when provided with the appropriate substrate, allowing for the detection and quantification of biotinylated biomolecules.

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Lab products found in correlation

Biotin anti cd28, by BD (2 mentions) Anti myc tag m0473, by MBL Life Science (2 mentions) Anti actin a5441, by Merck Group (2 mentions) Anti ptpn7 ab118978, by Abcam (2 mentions) Anti shp 1 ms1190, by Thermo Fisher Scientific (2 mentions) Anti pplc γ1 44 696g, by Thermo Fisher Scientific (2 mentions) Anti oxidized ptp active site mab mab2844, by R&D Systems (2 mentions) Anti flag f1804, by Merck Group (2 mentions) Magellan data analysis software, by Tecan (1 mentions) Tmb solution, by Abcam (1 mentions) 4 dimethylaminopyridine, by Merck Group (1 mentions) Penicillin streptomycin, by Corning (1 mentions) Protease inhibitor, by Thermo Fisher Scientific (1 mentions) Phosphate buffered saline (pbs), by Corning (1 mentions) 3 3 5 5 tetramethylbenzidine (tmb), by Merck Group (1 mentions) Piperidine, by Merck Group (1 mentions) 1 4 dioxane, by Merck Group (1 mentions) N hydroxysuccinimide, by Merck Group (1 mentions) 1 ethyl 3 3 dimethylaminopropyl carbodiimide, by Merck Group (1 mentions) Tetrabutylammonium bromide, by Merck Group (1 mentions)

Spelling variants of this product

Streptavidin-HRP (82 citations) Streptavidin–horseradish peroxidase (HRP) conjugate (9 citations) Streptavidin-HRP polymer conjugate (2 citations)

13 protocols using streptavidin hrp conjugate

Antibodies for Poliovirus Detection

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Mouse monoclonal anti-poliovirus VP3, 2B, 2C, and 3A, and rabbit polyclonal antibodies against poliovirus 3B were a gift from prof. Kurt Bienz (University of Bazel, Switzerland) and were partially described in [121 (link)–123 (link)]. Rabbit polyclonal anti-poliovirus 3D antibodies were custom generated by Chemicon and were described in [36 (link)]. Mouse monoclonal anti-dsRNA antibody J2 was from English & Scientific Consulting Kft.
Secondary Alexa dye fluorescent antibody and streptavidin conjugates were from Thermo Fisher, HRP secondary antibody conjugates were from Amersham or KPL. Streptavidin HRP conjugate was from Millipore Sigma (RPN1231).

Moghimi S., Viktorova E.G., Gabaglio S., Zimina A., Budnik B., Wynn B.G., Sztul E, & Belov G.A. (2022). A Proximity biotinylation assay with a host protein bait reveals multiple factors modulating enterovirus replication. PLOS Pathogens, 18(10), e1010906.

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Extracellular TG2 Activity Quantification

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Human pulmonary artery adventitial fibroblast cells were seeded in fibroblast growth media as described above on a 96-well cell culture plates (MilliporeSigma). Fibroblast growth media was replaced with reduced-serum fibroblast basal media (0.2% FBS). Next day, a TG2 substrate, 50µM biotin cadaverine (N-(5-Aminopentyl) biotinamide, trifluoroacetic acid Salt) (Cat# A1594; Thermo Fisher Scientific) was then added to the wells. After 24 hours, cell plates were washed with PBS and blocked with 1% BSA in PBS for 1 hour. Cells were then incubated with Streptavidin-HRP conjugate (Cat# GERPN1231; Millipore Sigma) in blocking buffer for 1 hour followed by washing with PBS. For extracellular TG2 activity, cultured cell plates were de-cellularized with 0.25M NH₄OH (MilliporeSigma) in 50mM Tris pH 7.4 for 10 minutes at room temperature as described previously (15 (link)). Cells were then incubated with a chromogenic substrate and visualizing reagent, TMB solution (Cat# ab171522; Abcam). After 5 minutes, Stop Solution for TMB (Cat# ab171529; Abcam) was added to each well and the 450nm absorbance was read in a Tecan SPECTRAFluor Plus microplate reader (Artisan Technology Group, Champaign, IL) equipped with Magellan Data Analysis software (Tecan).

Penumatsa K.C., Sharma Y., Warburton R.R., Singhal A., Toksoz D., Bhedi C.D., Qi G., Preston I.R., Anderlind C., Hill N.S, & Fanburg B.L. (2024). Lung-specific interleukin 6 mediated transglutaminase 2 activation and cardiopulmonary fibrogenesis. Frontiers in Immunology, 15, 1371706.

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Immune Receptor Signaling Pathway Analysis

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Stimulation: Biotin-anti-CD3 (553060), Biotin-anti-CD4 (553728), and Biotin-anti-CD28 (553296) were from BD Biosciences. Immunoprecipitation: anti-Flag (F1804) Sigma Aldrich; anti-SHP-1 (SC287), anti-SHP-2 (SC280) Santa Cruz. Western blotting: anti-pTyr (05321), anti-GST (06332), anti-ERK (06182), anti-SHP-1 (06117) EMD Millipore; anti-pTyr564-SHP-1 (8849), anti-pTyr416-Src (2101) Cell signaling Technology; anti-pTyr319-ZAP-70 (612574), anti-GRB2 (610111) BD Biosciences; anti-LCK (SC433), anti-ZAP-70 (SC574), anti-pERK (SC7383), anti-HA tag (SC805) Santa Cruz; anti-PTPN7 (ab118978) Abcam; anti-Myc tag (M0473) MBL International; anti-SHP-1 (MS1190) Thermo Fisher; anti-pTyr536-SHP-1(SP1571) ECM Biosciences; anti-actin (A5441) Sigma Aldrich; anti-pPLC-γ1 (44-696G) Biosource;, anti-Oxidized PTP active site mAb (MAB2844) R&D Systems. Rabbit polyclonal antisera to THEMIS^{7 (link)} and THEMIS2^{13 (link)} have been described. Streptavidin-HRP conjugate was purchased from Sigma Aldrich. Streptavidin was purchased from Southern Biotechnology.

Choi S., Warzecha C., Zvezdova E., Lee J., Argenty J., Lesourne R., Aravind L, & Love P.E. (2017). THEMIS enhances TCR signaling and enables positive selection by selective inhibition of SHP-1. Nature immunology, 18(4), 433-441.

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Immobilized TRAIL Binding Assay

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6× His-TRAIL was obtained from UBPBio. Mouse Anti-Human TRAIL-UNLB and mouse Anti-Human TRAIL-BIOT (Dallas, TX, USA) were purchased from SouthernBiotech (Birmingham, AL, USA) for ELISA. Dulbecco’s Modified Eagle Medium (DMEM), penicillin–streptomycin, fetal bovine serum (FBS), trypsin, and phosphate-buffered saline (PBS) were purchased from Corning (Corning, NY, USA). Heparin and Bacteroides Heparinase II were received from Selleckchem (Radnor, PA, USA) and New England Biolabs (Beverly, MA, USA), respectively. EZ-Cytox was purchased from DoGenBio (Seoul, Korea). Fmoc-Asp and Fmoc-Arg were obtained from BOC Science (Shirley, NY, US). Monomethoxy polyethylene glycol 750, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), N-hydroxy succinimide (NHS), tetrabutylammonium bromide (TBAB), 4-dimethyl amino pyridine (DMAP), dimethyl formamide (DMF) (anhydrous), 1,4-dioxane, piperidine, 3,3′,5,5′-Tetramethylbenzidine (TMB), and streptavidin-HRP conjugate were obtained from Sigma-Aldrich (St. Louis, MO, USA). Ethylene glycol diglycidyl ether (EGDE) was obtained from TCI (Tokyo, Japan). Protease inhibitor and Live and dead staining kit were obtained from ThermoScientific (Waltham, MA, USA).

Kim S., Jwa Y., Hong J, & Kim K. (2022). Inhibition of Colon Cancer Recurrence via Exogenous TRAIL Delivery Using Gel-like Coacervate Microdroplets. Gels, 8(7), 427.

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Calmodulin Protein Detection Protocol

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After SDS-PAGE and transfer, the resulting membrane was first dried and further incubated for 45 min in TBS buffer containing 0.1% (w/v) Triton X-100. The membrane was then incubated for 1 h in TBS buffer containing 0.1% (w/v) Triton X-100 and 5% (w/v) defatted milk followed by 1 h in saturation buffer containing 3% (w/v) gelatin, 50 mm Tris-HCl, pH 7.5, 50 mm MgCl₂, 150 mm NaCl, and 5 mm CaCl₂. Hybridization was then performed for 2 h in hybridization buffer containing 1% (w/v) gelatin, 50 mm Tris-HCl, pH 7.5, 50 mm MgCl₂, 150 mm NaCl, 5 mm CaCl₂, and 0.1% (w/v) Tween 20) + 0.1 μg/ml biotinylated CaM (Sigma). Detection of the bound CaM protein was then performed using a streptavidin–HRP conjugate (Sigma) and ECL using the manufacturer's instructions. Alternatively, after transfer, washing, and saturation, the membrane was incubated in the hybridization solution containing 0.3 μg/ml of a homemade CaM–HRP conjugate (the recombinant Arabidopsis CaM1 (AT5G37780) was covalently coupled to HRP). Detection of the bound CaM protein was then performed using ECL according to the manufacturer's instructions.

Moyet L., Salvi D., Bouchnak I., Miras S., Perrot L., Seigneurin-Berny D., Kuntz M, & Rolland N. (2019). Calmodulin is involved in the dual subcellular location of two chloroplast proteins. The Journal of Biological Chemistry, 294(46), 17543-17554.

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Western Blot Analysis of Proteins

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Proteins were separated by SDS-PAGE and transferred onto PVDF membranes (Thermofisher Cat.88518) via the Trans-Blot®Turbo™ System (Bio-Rad). The membranes were blocked in PBS containing 5 % bovine serum albumin (Sigma-Aldrich Cat.A7906) and 1 % Tween20 for 1 h at room temperature. Blots were probed with primary antibodies against beta-actin (mouse host; Sigma-Aldrich Cat.A2228) and biotin using the streptavidin-HRP conjugate (Sigma-Aldrich Cat. 18–152) in PBS containing 3 % bovine serum albumin and 1 % Tween20 at 4 °C overnight. The PVDF membranes were then washed three times with PBS containing 1 % Tween20 and probed with the secondary antibody anti-mouse IgG-HRP conjugate (Invitrogen™ Cat.62-6520) in PBS containing 3 % serum bovine albumin and 1 % Tween20 for 1 h at room temperature. The membranes were again washed three times with PBS 1 % Tween20 and kept in distilled water. Detection of the proteins on the membranes was performed with a chemiluminescence camera using the Pierce™ ECL Western Blotting Substrate (Thermo Scientific™ Cat. 32106). Quantification of signals on the membranes was done by densitometric scanning using the ImageJ software.

Luo T., Pueyo J.M., Wahni K., Yvanoff C., Lazar T., Pyr dit Ruys S., Vertommen D., Ezeriņa D, & Messens J. (2021). Thiol-disulphide independent in-cell trapping for the identification of peroxiredoxin 2 interactors. Redox Biology, 46, 102066.

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Screening sdAbs Binding to DIII Regions

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Purified sdAbs (1 μl) were spotted on NC membranes, and non-specific binding sites were blocked with 5% BSA dissolved in tris-buffered saline (TBS; pH = 7.3, Sigma-Aldrich). After a wash with TBS containing 0.05% Tween 20 (TBST-20; 5 min), membranes were separately incubated (1 h) with either biotinylated DIII-1^G299–K307 or DIII-2^V371–R388 reconstituted in a phosphate buffer (1 μg/ml, pH = 7.0). After three washings, streptavidin-HRP conjugate (1:30,000 in TBST-20; Sigma-Aldrich) was added, and the membranes were incubated for 1 h. Subsequently, five washes with TBST-20 (5 min each) and one with TBS (5 min) were performed before incubating the membranes with a SuperSignal West Dura chemiluminescent substrate, and the signal was recorded on a C-DiGit Blot Scanner (Odyssey CLx, Cambridge, United Kingdom). Simultaneously, sdAbs raised against N. meningitides [VHH_F3 (Kulkarni et al., 2020 (link)): non-related sdAbs; negative control] and hyperimmune serum of horse surviving natural WNV infection (positive control) spotted on NC membranes, were included in the assay to undergo incubations with DIII-1^G299–K307 or DIII-2^V371–R388, streptavidin-HRP conjugate, and chemiluminescent substrate, followed by signal detection.

Hruškovicová J., Bhide K., Petroušková P., Tkáčová Z., Mochnáčová E., Čurlík J., Bhide M, & Kulkarni A. (2022). Engineering the Single Domain Antibodies Targeting Receptor Binding Motifs Within the Domain III of West Nile Virus Envelope Glycoprotein. Frontiers in Microbiology, 13, 801466.

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Glycosylation Analysis of Recombinant Feline Interferon-α15

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Purified rFeIFN-α15 was digested with glycosidase F (Takara Bio, Shiga, Japan) at 37 °C for 17 h in the presence or absence of 0.5% SDS and 1.0% NP-40. The samples were separated on SDS-PAGE, followed by blotting to Immobilon-P membranes (Merck Millipore, Tokyo, Japan). For immunoblotting, the membranes were incubated with primary anti-FeIFN-α15 polyclonal rabbit antibody, generated by us, and secondary horseradish peroxidase (HRP)-conjugated anti-rabbit goat IgG (Sigma-Aldrich, Tokyo, Japan), with HRP detected with chemiluminescent reagents (ECL Western Blotting Detection System) (GE Healthcare). For lectin-blotting, the membranes were treated with biotinylated Con A (Seikagaku Corporation, Tokyo, Japan) and the resulting biotin residues reacted with streptavidin HRP conjugate (Sigma-Aldrich). HRP was detected with 3,3-diaminobenzidine (Wako Chemicals, Tokyo, Japan).

Minagawa S., Nakaso Y., Tomita M., Igarashi T., Miura Y., Yasuda H, & Sekiguchi S. (2018). Novel recombinant feline interferon carrying N-glycans with reduced allergy risk produced by a transgenic silkworm system. BMC Veterinary Research, 14, 260.

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Ascaris-Specific Antibody Response in EAH Mice

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Three experimental groups were formed (6 animals/group). EAH was induced in the mice by intravenously administering 20 mg/kg of ConA diluted in sterile PBS (2 mg/mL). After two hours, some of these animals received PBS (i.p.) (EAH group) and the remainder of the animals received 1 mg of ASC (i.p.) (EAH+ASC group). The control animals received only PBS (control group). The experimental model and hepatitis parameters were adjusted as described by Nascimento et al. (2014) (link). This protocol was approved by the Ethics Committee of the Biological Sciences Research Center, UFPE (no. 23076.041797/2013-40) .
Detection of anti-Ascaris antibodies using ELISA Blood samples were collected 8 h, 24 h and 7 days after induction of EAH. Ascaris-specific IgG1 and IgG2a antibodies were titrated using ASC (5 μg/mL)-coated 96-well plates (Nunc MaxiSorp, Denmark) and biotinylated goat anti-mouse IgG1 or IgG2a (Southern Biotechnology Associates Inc, USA). The reactions were developed using a streptavidin-HRP conjugate (Sigma-Aldrich, USA) and an O-phenylenediamine (Sigma-Aldrich, USA) solution in 0.1 M citrate buffer plus H 2 O 2 . The plates were read in 450 nm. The results were expressed as the mean of optical density ± standard deviation at a dilution corresponding to the linear part of the titration curve (1:10).

Silva R.C.P., Silva R.P.C.D., Silva M.D.C., Nascimento W.R.C.D., Costa V.M.A., Azevedo Albuquerque M.C.P, & Souza V.M.O. (2020). Extract of Ascaris suum induces TGF-β and early production of IgG1 in experimental autoimmune hepatitis. Revista brasileira de parasitologia veterinaria = Brazilian journal of veterinary parasitology : Orgao Oficial do Colegio Brasileiro de Parasitologia Veterinaria, 29(2).

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Dot Blot and Western Blot Analysis of Recombinant HCV Core Protein

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For dot blot analysis of proteins, equal concentration of TSP (5 µg-25 µg) from plant extracts, 5 µg from eHCV as positive control and 25 µg of non-agroinfiltrated plant extract were directly dotted on a nitrocellulose membrane. The membrane was dried at room temperature, blocked with 5% (w/v) skim milk in PBS-Tween (1/1000 v/v), and after three washing steps incubated with anti-C/N terminal 5xHis antibody (Qiagen) 1:10000 in TBS-T for two hours at room temperature. Following the washing steps, the membrane was incubated with 1:8000 diluted horseradish peroxidase (HRP)-conjugated goat anti-mouse antibodies (Sigma, USA) for one hour. Finally, 3-3’diaminobenzidine (DAB, Sigma, USA) was used for color development.
For SDS-PAGE and western blotting analyses, purified HCV core from E. coli and plant and plant crude extracts of the recombinant HCV core protein expressed by P19 co-agroinfiltrated pBI121 and PVX vectors were loaded onto a 12% SDS-polyacrylamide gel; by the end of electrophoresis, the protein bands were either stained with coomassie brilliant blue (Bio-Rad) or transferred to the PVDF membrane. Subsequently, the corresponding protein bands on the membrane were identified by biotinylated anti-core polyclonal antibody (Abcam, UK) (1:1000 dilution) and streptavidin HRP conjugate (Sigma, USA) (1:4000 dilution), respectively, and were detected by TMB as substrate.

Mohammadzadeh S., Khabiri A., Roohvand F., Memarnejadian A., Salmanian A.H., Ajdary S, & Ehsani P. (2014). Enhanced-Transient Expression of Hepatitis C Virus Core Protein in Nicotiana tabacum, a Protein With Potential Clinical Applications. Hepatitis Monthly, 14(11), e20524.

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