Strep tactin hrp conjugate

Manufactured by Bio-Rad

Sourced in United States

Strep Tactin-HRP conjugate is a laboratory reagent that contains Strep-Tactin, a modified form of the Streptavidin protein, conjugated to Horseradish Peroxidase (HRP). This conjugate is designed for use in various immunoassay and detection applications that involve biotin-streptavidin interactions.

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8 protocols using strep tactin hrp conjugate

Quantification of Kidney Organoid Proteins

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Kidney organoids were washed with PBS and lysed with 10mM Tris (pH 7.5) containing 1% sodium dodecyl sulfate (SDS) and 1 mM NaVO₄. Equal amounts of protein were separated on 7% SDS-PAGE gels and transferred to a polyvinylidene difluoride membrane (IB401001; Invitrogen, Carlsbad, CA, USA) using the iBlot2 Gel Transfer Device (IB21001; Invitrogen). Membranes were blocked with 5% skim milk in PBS with 0.1% Tween 20 detergent (T-PBS) for 1 h at room temperature and incubated with anti-NCC (ab224762, 1:500; abcam) and biotinylated Lotus Lectin (LTL, B-1323,1:100; Vector Laboratories, Burlingame, CA, USA), E-cadherin (ECAD, 610405, 1:500; BD Biosciences, San Jose, CA, USA), and b-actin (1:2000, 3700; Cell signaling Technology, Danvers, MA, USA) at 4 °C overnight. Membranes were then washed 3 times with T-PBS and incubated for 1 h with a goat anti-rabbit IgG-HRP conjugate (1706515, 1:1000; Bio-Rad, Hercules, CA, USA), a goat anti-mouse IgG-HRP conjugate (1706516, 1:1000; Bio-Rad), and a Strep Tactin-HRP conjugate (1610381, 1:1000; Bio-Rad). After 4 washes with T-PBS, the protein of interest was detected using an enhanced chemiluminescence system (WSE-7110, ATTO Corp., Tokyo, Japan). Quantification of relative densities was performed with the control group set at 100%; densities were normalized to those of b-actin bands from the same gel (Quantity One version 4.4.0; Bio-Rad).

Lim S.W., Fang X., Cui S., Lee H., Shin Y.J., Ko E.J., Lee K.I., Lee J.Y., Chung B.H, & Yang C.W. (2023). CRISPR-Cas9-Mediated Correction of SLC12A3 Gene Mutation Rescues the Gitelman’s Disease Phenotype in a Patient-Derived Kidney Organoid System. International Journal of Molecular Sciences, 24(3), 3019.

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Immunohistochemical Analysis of CD3+ T Cells

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Fixed eyeballs were dehydrated, paraffin embedded, and sectioned. Slides with the sections were stained with rabbit anti-mouse CD3 (1:500; R&D Systems, Minneapolis, MN), followed by incubation with rabbit antibody conjugated with Biotin (Jackson Laboratories, Bar Harbor, ME) was added and incubated for 1 hour. Strep-Tactin-HRP Conjugate (Bio-Rad Laboratories) at 1:500 dilution was used to amplify the signals. The substrate solution including 3,3′-diaminobensidine was added for visualization of positive cells by light microscopy.

Yang J., Park J.W., Zheng D, & Xu R.H. (2018). Universal Corneal Epithelial-Like Cells Derived from Human Embryonic Stem Cells for Cellularization of a Corneal Scaffold. Translational Vision Science & Technology, 7(5), 23.

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Antibody Characterization for Inflammation

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Rabbit polyclonal anti-human COX-2, rabbit polyclonal anti-phospho-5-Lypoxygenase (5-LO) and cPLA₂ rabbit polyclonal antibodies (Abs), were purchased from Cayman Chemical (CA, USA), R&D (MN, USA), and Cell Signaling (MA, USA), respectively. Rabbit monoclonal anti-mouse COX-2 monoclonal Abs, mAbs (clone SP21) was purchased from Thermo Fisher Scientific (MA, USA). Unconjugated mouse anti-human COX-2 mAbs (clone 33) was purchased from BD (CA, USA) and labeled using Zenon Mouse IgG labeling kit was purchased from Invitrogen (CA, USA). Rabbit monoclonal anti-β-actin mAbs (clone RM112) was purchased from Millipore (MS, USA). Precision plus Dual Color standard, Western C standard, and StrepTactin-HRP Conjugate were purchased from BioRad, (CA, USA.). Fluorochrome-conjugated murine anti–α-smooth muscle actin (α-SMA; clone 1A4) monoclonal mAb was purchased from Sigma-Aldrich (St. Louis, MO).

Uribe G., Villéger R., Bressollier P., Dillard R.N., Worthley D.L., Wang T.C., Powell D.W., Urdaci M.C, & Pinchuk I.V. (2018). Lactobacillus rhamnosus GG increases COX 2 expression and PGE2 secretion in colonic myofibroblasts via a MyD88 dependent mechanism during homeostasis. Cellular microbiology, 20(11), e12871.

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Western Blot Analysis of Protein Targets

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Protein was extracted using Pierce RIPA buffer (Thermo Scientific) and protease inhibitor (Thermo Scientific). The samples were quantified by BCA assay (Thermo Scientific). 20ug of protein was mixed with 2X laemmeli buffer and BME for a 1:1 sample, denatured at 56°C for five minutes and electrophoresed and transferred with a molecular marker according to the manufacturer’s protocol (Mini-Protean Tetra Cell, BioRad). Membranes were stained with 0.1% Ponceau S to confirm sufficient protein transfer. The membrane was blocked in 10 mL of 5% Non-Fat dry milk in 1X TBS-T for 1 hour, followed by three 15-minute washes with 1X TBS-T. Blots were incubated in primary antibody (COX2 1:500 Protein tech #663511Ig, ECAD 1:3,000 Protein Tech #20874-1-AP) overnight at 4°C. Membranes washed and incubated in diluted 1:5,000 secondary anti-rabbit IgG antibody (BioRad) and 1:5000 StrepTactin HRP conjugate (Biorad) for 1 hour. Membranes were washed and exposed to 2 mL of Clarity Western ECL substrate (Biorad) for five minutes. Images were captured and quantified with a ChemiDoc imager (BioRad). To confirm protein loading, the membrane was stripped, rinsed, blocked, and probed for B-actin (Biorad #MCA5775GA).

Sompel K., Dwyer-Nield L.D., Smith A.J., Elango A., Backos D.S., Zhang B., Gross J., Ternyak K., Matsuda J.L., Kopf K., Keith R.L, & Tennis M.A. (2022). Iloprost requires the Frizzled-9 receptor to prevent lung cancer. iScience, 25(6), 104442.

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PilJ Protein Expression Analysis in P. aeruginosa

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P. aeruginosa strains with C-terminally His-tagged PilJ at the native site in the chromosome were incubated overnight in LB with aeration at 37°C, subcultured 1:100 in LB the following morning, then standardized to OD₆₀₀ 0.01 in LB before spread plating 100 μL on LA. Plates were incubated overnight at 37°C. The following day, cells were scraped up in 1 mL of phosphate buffered saline (PBS), pelleted and washed once in PBS, then resuspended in 1 mL PBS. The OD₆₀₀ for each cell suspension was measured, and an aliquot of 1 mL of cells at OD₆₀₀ 10 were pelleted on ice. Pellets were resuspended in RIPA buffer (50 mM Tris (pH 7.9), 150 mM NaCl, 1% NP-40, 0.5% Tween-20, 0.1% SDS) and briefly sonicated on ice to lyse cells. Proteins were standardized to 25 ug, following a bicinchoninic acid (BCA) assay, run on a 4% to 15% Tris-glycine polyacrylamide gel, then transferred onto a PVDF membrane. The membrane was blocked with 10% skim milk, and PilJ proteins were detected by chemiluminescence via incubation with primary mouse anti-His antibodies (Thermo Fisher), followed by incubation with goat-anti mouse, HRP (Thermo Fisher). The loading control was detected by incubation with StrepTactin-HRP conjugate (Bio-Rad). Blots were imaged using an Azure Sapphire.

Yarrington K.D., Shendruk T.N, & Limoli D.H. (2024). The type IV pilus chemoreceptor PilJ controls chemotaxis of one bacterial species towards another. PLOS Biology, 22(2), e3002488.

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Exosome Protein Detection Assay

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Exosomes and A549 cells transiently transfected with viral plasmids were lysed with RIPA buffer. One μg of lysate was separated by 8%–15% SDS-PAGE and transferred to nitrocellulose membranes (LI-COR Biosciences) and blocked with 5% BSA in PBS at 4°C overnight. Strep fusion protein and CD63 were visualized by Strep-Tactin-HRP conjugate (Bio-Rad) and Alexa Fluor fluorescent-conjugated anti-CD63 antibody, respectively.

Teng Y., Xu F., Zhang X., Mu J., Sayed M., Hu X., Lei C., Sriwastva M., Kumar A., Sundaram K., Zhang L., Park J.W., Chen S.Y., Zhang S., Yan J., Merchant M.L., Zhang X., McClain C.J., Wolfe J.K., Adcock R.S., Chung D., Palmer K.E, & Zhang H.G. (2021). Plant-derived exosomal microRNAs inhibit lung inflammation induced by exosomes SARS-CoV-2 Nsp12. Molecular Therapy, 29(8), 2424-2440.

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Western Blot Quantification Protocol

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Samples of cell lysates saved after TCA-precipitation and re-suspension (inputs) were analyzed by Western blotting. After standard SDS-PAGE, proteins were transferred to nitrocellulose membranes and stained with Ponceau S (Sigma Aldrich) prior to blocking to monitor equal protein loading. Membranes were blocked with 4% (w/v) non-fat milk powder in TBST (0.1% Tween-20 in Tris-buffered saline) at room temperature for 2 hours. Blocked membranes were incubated with StreptActin-HRP conjugate (Bio-Rad) diluted 1:50,000 in TBST with 4% non-fat milk powder overnight at 4 °C with constant shaking. Prior to chemiluminesce nt detection, blots were washed with TBST four times, 5 minutes each time. Western blots were developed using Amersham ECL Western Blotting Detection Kit (GE Healthcare).

Paek J., Kalocsay M., Staus D.P., Wingler L., Pascolutti R., Paulo J.A., Gygi S.P, & Kruse A.C. (2017). Multidimensional tracking of GPCR signaling via peroxidase-catalyzed proximity labeling. Cell, 169(2), 338-349.e11.

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Bovine Mastitis Immune Response Profiling

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Western blot was conducted as described by Abdi et al. [33 (link)]. Briefly, the SUSP were separated by electrophoresis and transferred to nitrocellulose membrane (Bio-Rad) at a constant 100 V with 400 mA for 60 min by a wet method (Bio-Rad). The membrane was blocked overnight, washed, and incubated with hyperimmune serum from a cow previously vaccinated with SUSP and protected from mastitis upon challenge in another previous pilot study. The membrane was washed and incubated with horse radish peroxidase (HRP)-conjugated sheep anti-bovine IgG (H+L) secondary antibody (Bethyl Lab. Montgomery, TX, USA). The precision protein Strep Tactin-HRP conjugate (Bio-Rad) was used as a secondary antibody for molecular weight markers. Finally, the membrane was washed and 25 mL of peroxidase substrate (TMB membrane HRP substrate (SeraCare Life Sciences Inc, Milford, MA, USA) was added, and the reaction was fully developed at room temperature. Protein band images were taken using a ChemiDoc™ Touch Imaging System (Bio-Rad) and analyzed using Image Lab Software (Bio-Rad). Images on the gel and the membrane were compared to identify immune-reactive protein bands.

Kerro Dego O., Almeida R., Ivey S, & Agga G.E. (2021). Evaluation of Streptococcus uberis Surface Proteins as Vaccine Antigens to Control S. uberis Mastitis in Dairy Cows. Vaccines, 9(8), 868.

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