Streptavidin hrp conjugate

Manufactured by Thermo Fisher Scientific

Sourced in United States

Streptavidin-HRP conjugate is a protein complex composed of streptavidin, a tetrameric protein that binds biotin, and horseradish peroxidase (HRP), an enzyme that catalyzes chromogenic or chemiluminescent reactions. This conjugate is commonly used in various immunoassay techniques, such as ELISA, to detect and quantify biotinylated molecules.

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Streptavidin-HRP (301 citations) Streptavidin horseradish peroxidase (HRP) conjugate (15 citations) PierceTM High Sensitivity Streptavidin-HRP conjugate (3 citations) PierceTM High Sensitivity Streptavidin-horseradish peroxidase (HRP) conjugate (3 citations) High-sensitivity streptavidin-HRP conjugate (2 citations) Stabilized Streptavidin-HRP Conjugate (2 citations) Streptavidin-HRP conjugate (SA-HRP; (2 citations)

49 protocols using streptavidin hrp conjugate

Profiling Papain-Like Proteases in Citrus

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Papain (Sigma-Aldrich), Nicotiana bethamiana apoplastic fluids, and total citrus leaf extracts were pretreated with either buffer control, E-64, or SDE1 recombinant proteins. Total leaf extracts from SDE1-expressing transgenic citrus lines were pretreated with either 100 μM E-64 or buffer control. After pretreatment, the samples were incubated with a final concentration of 2 μM DCG-04^{24 (link)} for 4 h at room temperature, followed by precipitation with 100% ice-cold acetone. Samples were centrifuged at 12,000×g, washed with 70% acetone, then centrifuged again. Precipitated products were re-suspended in 50 mM Tris buffer (pH 6.4) and either used directly for western blotting using Streptavidin-HRP conjugates (Thermo Scientific) or further enriched on streptavidinmagnetic beads (Thermo Scientific). For enrichment, samples were incubated with 25 μL streptavidin magnetic beads at room temperature for 1 hr, washed twice with 1% SDS, and eluted by heating for 5 min at 95 °C in Laemmli sample buffer with 13% β-mercaptoethanol^{50 (link)}. The labeled proteins were separated using SDS-PAGE and active proteases were visualized by western blotting using Streptavidin-HRP conjugates (Thermo Scientific).
The experiments were repeated two times with similar results. Uncropped raw data are presented in Supplementary Fig. 11.

Clark K., Franco J.Y., Schwizer S., Pang Z., Hawara E., Liebrand T.W., Pagliaccia D., Zeng L., Gurung F.B., Wang P., Shi J., Wang Y., Ancona V., van der Hoorn R.A., Wang N., Coaker G, & Ma W. (2018). An effector from the Huanglongbing-associated pathogen targets citrus proteases. Nature Communications, 9, 1718.

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Quantifying Progranulin Levels in iPSCs

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We coated high-binding 96-well enzyme immunoassay/radioimmunoassay plates (Corning) overnight with a monoclonal antibody raised against the C-terminus of human progranulin (PGRN; 1.5 µg/ml; a gift of Laura Mitic, Bluefield Project, San Francisco, CA) and blocked with 1% BSA for 1 h at 37°C. We then incubated plates with conditioned medium collected for 24 h from GRN^+/+ and GRN^−/− iPSCs or recombinant human PGRN (0–32 ng/ml, R&D Systems) for 1 h at 37°C. We then incubated plates with an N-terminal monoclonal PGRN antibody (1.5 µg/ml; a gift of Laura Mitic), anti-mouse biotinylated IgG (1:5,000, Vector Laboratories), and streptavidin-HRP conjugate (1:10,000, Thermo Fisher Scientific). We developed the reactions at room temperature using 3,3′,5,5′-tetramethylbenzidine substrate (Thermo Fisher Scientific), quenched reactions with 1 N HCl, and performed analysis at 450 nm on a SpectraMax M5 spectrophotometer (Molecular Devices).

Narendra D.P., Guillermier C., Gyngard F., Huang X., Ward M.E, & Steinhauser M.L. (2019). Coupling APEX labeling to imaging mass spectrometry of single organelles reveals heterogeneity in lysosomal protein turnover. The Journal of Cell Biology, 219(1), e201901097.

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Western Blot Analysis of Cellular Proteins

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Cells were lysed with RIPA buffer (Sigma) and centrifuged at 13000 rpm for 15 min in 4°C. Proteins were resolved on SDS/PAGE and transferred onto a nitrocellulose membrane. The membrane was then blocked with 3% bovine serum albumin (BSA) in tris-buffered saline, with tween-20 (TBST) for 30 min at RT, followed by primary antibody incubation with mouse anti-PAC (1:200), mouse anti-FLAG (1:1000) or rabbit anti-GAPDH (1:1000; CST) at 4°C for overnight. After wash, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibody for 1 h at RT. Immunoblot samples from cell-surface biotinylation were blocked in 5% BSA and then incubated with streptavidin HRP-conjugate (1:5000, Thermo Scientific) for 1 h. Proteins were then detected using Pierce ECL Plus Western Blotting Substrate (Thermo Scientific) and quantified using ImageJ.

Osei-Owusu J., Yang J., Leung K.H., Ruan Z., Lü W., Krishnan Y, & Qiu Z. (2021). Proton-activated chloride channel PAC regulates endosomal acidification and transferrin receptor-mediated endocytosis. Cell reports, 34(4), 108683.

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Detailed Reagents for Cell Culture

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Dulbecco’s modified minimal essential medium (DMEM), fetal bovine serum (FBS), phosphate-buffered saline (PBS), trypsin-EDTA solution, 100× penicillin–streptomycin–l-glutamine solution, and streptavidin-HRP conjugate were purchased from Thermo Fisher (Waltham, MA). Puromycin and G418 solution were obtained from InvivoGen (San Diego, CA). Hygromycin B solution was purchased from Enzo Life Science (Farmingdale, NY). Anti-ORF57, anti-K8, and anti-K8.1 antibodies were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Anti-K-Rta antibody was described previously (55 (link)). All other chemicals were purchased from Millipore-Sigma (St. Louis, MO) unless otherwise stated.

Kumar A., Salemi M., Bhullar R., Guevara-Plunkett S., Lyu Y., Wang K.H., Izumiya C., Campbell M., Nakajima K.I, & Izumiya Y. (2021). Proximity Biotin Labeling Reveals Kaposi’s Sarcoma-Associated Herpesvirus Interferon Regulatory Factor Networks. Journal of Virology, 95(9), e02049-20.

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Quantification of Human TNFR2 Binding to Biotinylated TNFα

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Human TNFα (Gibco, PHC3011) was biotinylated using an EZ-Link™ Micro Sulfo-NHS-Biotinylation Kit (ThermoFisher Scientific, 21925) according to manufacturer’s instructions. Human TNFR2 (Acro Biosystem, TN2-H5227) was coated on high binding polystyrene flat bottom micro-titer plates (Thermo Scientific, 3455). Plates were incubated at 4°C overnight. Plates were washed with PBST (wash buffer 0.1%) and blocked with 1% BSA (Fisher Scientific, BP1600-100) in PBS for 1 hour at 37°C. AN3025 or human IgG₁, κ (BioxCell, BE0297) was titrated, distributed 50μL per well and incubated for 1 hour at 37°C. 50μL biotinylated human TNFα was added per well to achieve the final TNFα concentration at 100ng/ml. After another 1-hour incubation, plates were washed with PBST (wash buffer 0.1%). Streptavidin-HRP conjugate (Thermo Scientific, N504) was distributed to detect bound biotinylated human TNFα. After 1-hour incubation of secondary antibody at 37°C, plates were washed with PBST (wash buffer 0.1%). Plates were developed using TMB substrate solution (eBioscience,00-4201-56) and stopped with ELISA stop solution (Invitrogen, SS04). The level of bound biotinylated human TNFα was determined by reading absorbance at 450 nm.

Chen Y., Jia M., Wang S., Xu S, & He N. (2022). Antagonistic Antibody Targeting TNFR2 Inhibits Regulatory T Cell Function to Promote Anti-Tumor Activity. Frontiers in Immunology, 13, 835690.

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Affinity Purification and EMSA Analysis

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I-SceI and ISVB2 were tagged at the N-terminus with a GST epitope and were then purified using a GST agarose column (GE healthcare). DNA probes with 3′-biotin-labeled were ordered directly from Invitrogen. We followed the protocol developed by Ruff et al. (23 (link)), in which double-stranded DNA probes (5 nmol) were mixed with various concentrations of either GST-I-SceI or GST-ISVB2 in EMSA reaction buffer (20 mM Tris-HCl, pH 8.5, 50 mM NaCl, 2 μM ZnCl₂, 12 mM MgCl₂, 2% glycerol, 2 mg/ml BSA and 2 mM freshly prepared DTT). All reaction mixtures were incubated at 25°C for 40 min and subsequently analyzed on 4% non-denaturing polyacrylamide gels. Samples in the gels were then transferred to nylon membranes (Milipore) and ultraviolet-crosslinked at 2000 J. The crosslinked membranes were blocked in 5% non-fat milk, followed by a 1-h incubation in 1:10 000 diluted streptavidin-HRP conjugate (Thermo) and then washed three times. The chemiluminescent signal was detected using the chemiluminescence labeling detection reagent ECL Plus (GE healthcare) and analyzed using the Bio-Rad Chemidoc XRS+. The I-SceI and β-globin probes had the following sequences: 5′-TGCACCATTCTTAGGGATAACAGGGTAATTTTCTGGGTTA-3′ and 5′-TGCACCATTCTAAAGAATAACAGTGATAATTTTCTGGGTTA-3′, respectively.

Lin J., Chen H., Luo L., Lai Y., Xie W, & Kee K. (2014). Creating a monomeric endonuclease TALE-I-SceI with high specificity and low genotoxicity in human cells. Nucleic Acids Research, 43(2), 1112-1122.

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Bacterial Specificity of Phage-Displayed Peptide

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To analyze the specificity of the peptide identified by phage display, the peptide was subjected to ELISA against different bacteria as described in Section 2.4. The bacterial cells were resuspended in PBS and incubated in ELISA plates overnight at 4 °C to achieve coating. Subsequently, ELISA was performed using the biotinylated KP peptide (10 μM) as the recognition element. The bound peptide was detected using the streptavidin-HRP conjugate (1:2000 in blocking buffer, Thermo Fisher Scientific).

Kim H., Jang J.H., Jung I.Y, & Cho J.H. (2022). A Novel Peptide as a Specific and Selective Probe for Klebsiella pneumoniae Detection. Biosensors, 12(3), 153.

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Immunoprecipitation and Northern Blotting

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Nuclear extract from 1.0 × 10⁷ cells expressing FLAG-Chtop was used for this assay. BT-RNA (1 pmol) was added to the extract and subjected to immunoprecipitation using anti-FLAG magnetic beads as described in Supplementary Data. RNA isolated from FLAG-tagged protein complexes was subjected to denaturing urea-PAGE and Northern blotting (9 ,12 (link)). BT-RNA was detected with stabilized Streptavidin-HRP Conjugate (Thermo Scientific, 89880D). Chemiluminescence was detected with an ImageQuant LAS4000.

Izumikawa K., Yoshikawa H., Ishikawa H., Nobe Y., Yamauchi Y., Philipsen S., Simpson R.J., Isobe T, & Takahashi N. (2016). Chtop (Chromatin target of Prmt1) auto-regulates its expression level via intron retention and nonsense-mediated decay of its own mRNA. Nucleic Acids Research, 44(20), 9847-9859.

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Western Blot Analysis of Ion Channels

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Forty micrograms of total protein lysates were resolved by SDS-PAGE, transferred onto nitrocellulose membranes and blocked with BSA. The membranes were subsequently incubated overnight at 4°C in blocking buffer with the corresponding primary antibody: Ca_vβ_2b (1:250, homemade), Ca_vβ₂ (1:1,000, Novus Biologicals, NBP1 86680), Ca_v1.2 (1:500, Alomone Labs, ACC-003), RyR2 (1:1,000, Thermo Fisher Scientific, MA3-916), V5 epitope tag (1:1,000, Cell Signaling, #13203), SERCA2a (1:1,000, Thermo Fisher Scientific, MA3-919) and GAPDH (1:5,000, Cell Signaling, #2118). Afterwards, the membranes were washed with TBS-Tween and incubated with anti-rabbit IgG or anti-mouse IgG secondary antibodies conjugated to horseradish peroxidase (HRP) for 1 h at RT. For the biotinylated proteins, the membranes were incubated with streptavidin-HRP conjugate (1: 2,000, Thermo Fisher Scientific) during 2 h at RT. After washing, all the membranes were developed using the PierceTM ECL Western Blotting Substrate (Thermo Fisher Scientific).

Cruz-Garcia Y., Barkovits K., Kohlhaas M., Pickel S., Gulentz M., Heindl C., Pfeiffer K., Eder-Negrin P., Maack C., Marcus K., Kuhn M, & Miranda-Laferte E. (2022). Nanoenviroments of the β-Subunit of L-Type Voltage-Gated Calcium Channels in Adult Cardiomyocytes. Frontiers in Cell and Developmental Biology, 9, 724778.

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Synthesis and Characterization of PLGA-DAT

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Diamino tetraiodothyroacetic acid (DAT), deuterium-labelled DAT-D7 as internal standard, and DAT-conjugated PLGA polymer (PLGA-DAT) were synthesized by DPx Fine Chemicals (Regensburg, Germany). PLGA (average MW 8,000 g/mol), lactide:glycolide (79:21), Cremophor EL, acetonitrile (>99%), DMSO (99.9%), ethyl acetate (99.8%), poly (vinyl alcohol) (PVA, hydrolysed 88%, average MW 31,000 g/mol), deoxycholic acid (DCA, >99%), formic acid (>99.5%), and bovine serum albumin (BSA) were purchased from Sigma-Aldrich (St. Louis, MO) and used without further purification unless otherwise noted. Freeze-drying was performed using a Millrock Tech MD85 Console Manifold Freeze Dryer (Millrock Technology, Kingston, NY). Deionized water with resistivity of 18.0 MΩ was used in all experiments. Purified αvβ3 and anti-αvβ3 conjugated with biotin were obtained from Bioss Inc (Woburn, MA), streptavidin HRP conjugate were from Thermo Fisher Scientific (Grand Island, NY), fibrinogen was from Millipore Sigma (Burlington, MA), Cy5-NHS dye was from Lumiprobe (Hunt Valley, MD), and 3,3′,5,5′-tetramethylbenzidine (TMB) and TMB-stop solution were from ABCAM Inc (Cambridge, MA). Blank mouse plasma for the PK study of PLGA-DAT versus DAT was from BioreclamationIVT (Westbury, NY).

Li W., Yalcin M., Bharali D.J., Lin Q., Godugu K., Fujioka K., Keating K.A, & Mousa S.A. (2019). RETRACTED ARTICLE: Pharmacokinetics, Biodistribution, and Anti-Angiogenesis Efficacy of Diamino Propane Tetraiodothyroacetic Acid-conjugated Biodegradable Polymeric Nanoparticle. Scientific Reports, 9, 9006.

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