Protein lysate was run on a 4–20% Mini-Protean TGX gel (BioRad) and transferred onto a nitrocellulose membrane (BioRad). Membranes were incubated overnight at 4 °C with purified anti-MyD88 antibody (ProSci) followed by peroxidase-conjugated donkey anti-rabbit (Jackson ImmunoReseach) and Precision Protein StrepTactin-HRP Conjugate (BioRad). SuperSignal West Pico Chemiluminescent Substrate was used for imaging (Thermo Scientific). Membranes were stripped and incubated overnight at 4 °C with purified anti-β-actin (Invitrogen) followed by peroxidase-conjugated donkey anti-mouse (Jackson ImmunoResearch) and Precision Protein StrepTactin-HRP Conjugate (BioRad).
Precision protein streptactin hrp conjugate
Manufactured by Bio-Rad
Sourced in United States
Precision Protein StrepTactin-HRP Conjugate is a laboratory reagent used in various protein detection and purification methods. It consists of StrepTactin, a streptavidin-derived protein, conjugated to Horseradish Peroxidase (HRP) enzyme. This conjugate facilitates the specific detection and visualization of proteins tagged with the Strep-tag® II affinity peptide.
Automatically generated - may contain errors
Lab products found in correlation
Precision plus protein westernc standard, by Bio-Rad (3 mentions)
Precision plus protein westernc, by Bio-Rad (2 mentions)
Anti mouse antibody against β tubulin, by Merck Group (2 mentions)
Nitrocellulose membrane, by Bio-Rad (2 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (2 mentions)
Pvdf membrane, by Bio-Rad (2 mentions)
Trans blot turbo transfer system, by Bio-Rad (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Chemidoc analyzer, by Bio-Rad (1 mentions)
Trans blot turbo, by Bio-Rad (1 mentions)
Polyclonal donkey anti rabbit igg hrp, by Abcam (1 mentions)
Supersignal west dura extended duration substrate, by Thermo Fisher Scientific (1 mentions)
Precast gel, by Bio-Rad (1 mentions)
Molecular imaging software, by Kodak (1 mentions)
Ecl reagent, by GE Healthcare (1 mentions)
Chemidoc xrs plus imaging system, by Bio-Rad (1 mentions)
Laemmli sample buffer, by Thermo Fisher Scientific (1 mentions)
Image lab software, by Bio-Rad (1 mentions)
Anti β actin, by Thermo Fisher Scientific (1 mentions)
Mini protean tgx gel, by Bio-Rad (1 mentions)
25 protocols using precision protein streptactin hrp conjugate
1
Western Blot for MyD88 and β-Actin
2
Confirmation of Protein Expression by Western Blot
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The expression of each WT-HBc, ΔHBc or ZHER2-ΔHBc monomer was confirmed by western blotting. The purified WT-HBc, ΔHBc and ZHER2-ΔHBc particles were analysed by SDS-PAGE and electro-transferred onto a nitrocellulose membrane. For the detection of the His6-tag, rabbit anti-6-His antibody (Bethyl Laboratories, USA) was used as a primary antibody at 1:1000 dilution for immunoblotting, followed by HRP-linked anti-rabbit (Cell Signalling Technology, USA) at 1:1000 dilution and Precision Protein StrepTactin-HRP Conjugate (Bio-Rad Laboratories, USA) at 1:10,000 dilution for secondary antibody. For the detection of the His6-tag, mouse anti-HBc antibody (Merck Millipore, USA) was used as a primary antibody at 1:1000 dilution for immunoblotting, followed by HRP-linked anti-mouse (Cell Signalling Technology, USA) at 1:1000 dilution and Precision Protein StrepTactin-HRP Conjugate (Bio-Rad Laboratories, USA) at 1:10,000 dilution for secondary antibody. The specific bands were detected with enhanced chemiluminescence (ECL) detection system. The membrane was imaged using the ChemiDoc™MP (Bio-Rad Laboratories, USA) and analysed with Image Lab (Bio-Rad Laboratories, USA) software.
3
Western Blot Analysis of Protein Expression
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Habenular and striatal lysates were prepared as described for PKA enzymatic activity assays. Per lane, 10 μg of total protein was loaded onto 4%–12% Bis-Tris gels (Bolt Plus, Invitrogen, Thermo Fisher Scientific) and run for 35 minutes at 165 V. For each gel, 7 μL of WesternSure prestained protein ladder was loaded onto a separate lane (LI-COR Biosciences). Protein was transferred onto nitrocellulose membranes using a semidry apparatus for 30 minutes (TransBlot Turbo, Bio-Rad), stained with Ponceau S stain, washed with 1× TBS with 0.1% Tween-20 (1× TBST), and then blocked with 5% nonfat dry milk or bovine serum albumin in 1× TBST for 1 hour at room temperature. Membranes were then probed overnight with primary antibodies with gentle shaking at 4°C before washing 3 times with 1× TBST and probing for 1 hour at room temperature with the appropriate antibody and Precision Protein StrepTactin-HRP Conjugate (1:10,000, Bio-Rad). All Western blots were visualized using Pierce enzyme chemiluminescent substrate (Thermo Fisher Scientific) and a ChemiDoc analyzer (Bio-Rad). See Supplemental Table 1 for antibodies used, sources, and dilutions used.
4
Quantitative Immunoblotting of MMP7
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Immunoprecipitated proteins were subjected to polyacrylamide gel electrophoresis using precast gels (BioRad Laboratories Inc) and a standard preparation Laemmli buffer along with Precision Plus Protein WesternC Standards (BioRad Laboratories Inc). Gel was transferred overnight to nitrocellulose membrane, followed by blocking with 5% nonfat milk, 1% BSA, in PBS for 1 hour. The membrane was then probed with polyclonal rabbit anti-MMP7 (Abcam; Ab38999) primary antibody diluted in PBS-0.05% Tween 20 at 4°C overnight. Polyclonal donkey anti-rabbit IgG-HRP (Abcam) was used as a secondary antibody, along with Precision Protein StrepTactin-HRP Conjugate (BioRad Laboratories Inc) for 1 hour at room temperature. After washing of the membrane, SuperSignal West Dura Extended Duration Substrate (Thermo Fisher Scientific Inc) was used for chemiluminescent detection of probed proteins, visualized with a Kodak Gel Logic 100 system (Eastman Kodak, Rochester, NY). Blot band strengths were quantified using Kodak Molecular Imaging Software (Eastman Kodak) and normalized to baseline uninjured tissue protein levels.
5
Western Blot Analysis of Vero Cell Proteins
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Protein extracts from Vero cells were separated by electrophoresis in 12% acrylamide–bisacrylamide gels, or 7% to detect PIKfyve. Separated proteins were transferred to nitrocellulose membranes, and proteins were detected with specific antibodies in each case. As a secondary antibody, anti-mouse IgG (GE Healthcare, Little Chalfont, UK) or anti-rabbit IgG (Bio-Rad, Oslo, Norway) conjugated to horseradish peroxidase, were used at a 1:5000 dilution. Precision Protein StrepTactin-HRP Conjugate (Bio-Rad) was used to reveal the ladder Precision Plus Protein WesternC (Bio-Rad). As a load control in WB analysis, an anti-mouse antibody against β-tubulin (Sigma) 1:2000 was used. Finally, bands obtained after development with ECL reagent were detected on the Molecular Imager Chemidoc XRSplus Imaging System (Bio-Rad, Hercules, CA, USA). Bands were quantified by densitometry, and data were normalized to control values using Image system software.
6
Western Blot Analysis of BAG3 Protein
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Cells were harvested in Laemmli sample buffer (Life technologies), heated for 5 min at 95 °C and resolved by SDS-PAGE in 12% polyacrylamide-bisacrylamide gels. Afterwards, separated proteins were transferred to a nitrocellulose membrane (Bio-Rad, Hercules, CA, USA) and the non-specific antibody binding sites were blocked with skimmed milk diluted in PBS and then incubated with the specific primary and HRP (Horseradish peroxidase)-conjugated secondary antibodies. Antibodies used for western blotting included: anti-rabbit polyclonal against BAG3 1:500 (Proteintech). As a loading control an anti-mouse antibody against β-tubulin (Sigma) 1:2000 was used. As secondary antibodies, anti-mouse IgG (GE Healthcare, Piscataway, NJ, USA) or anti-rabbit IgG (Bio-Rad) conjugated to horseradish peroxidase were used at a 1:5000 dilution. Precision Protein StrepTactin-HRP Conjugate (Bio-Rad) was used to reveal the ladder Precision Plus Protein WesternC (Bio-Rad). Finally, bands obtained after development with ECL reagent (GE Healthcare, Piscataway, NJ, USA) were detected using a Chemidoc XRSplus Imaging System (Bio-Rad). Band densitometry was performed with Image Lab software (Bio-Rad Inc: 2011) and data were normalized to loading control values.
7
Western Blot Analysis of Tagged Proteins
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Proteins were separated by NuPAGE 4–12% Bis-Tris gels (ThermoFisher Scientific) and transferred to nitrocellulose membranes (Amersham Protran Premium, 0.45 µm). The membranes were blocked with 5% nonfat dry milk (His6 tagged proteins) or 5% BSA (TwinStrep tagged proteins) in PBS for 3 h at room temperature. For His6 tag detection, the membranes were incubated with primary antibody (Penta-His Antibody, Qiagen, cat. no. 34660, dilution 1:1000) for 1 h at room temperature, washed three times for 10 min with PBS and incubated for 1 h at room temperature with secondary antibody conjugated to horseradish peroxidase (Anti-mouse IgG peroxidase polyclonal goat antibody, Sigma, cat. no. A0168, dilution 1:10,000). For TwinStrep tag detection, the membranes were incubated with antibody conjugated to horseradish peroxidase (Precision Protein StrepTactin-HRP Conjugate, BioRad, cat. no. 1610380, dilution 1:1000) for 1 h at room temperature. After washing three times for 10 min with PBS, the signal was detected using ECL (BioRad). Uncropped images of all western blots are shown in the Source data file.
8
Quantitative Western Blot Assay for AHA
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Cell lysate or protein extracts obtained from sucrose gradient fractions were supplemented with Pierce Lane Marker Reducing Sample Buffer (Thermo Scientific #39000), heated at 95°C for 10 min, and separated by SDS-PAGE. Separated proteins were transferred to nitrocellulose membranes, then membranes were blocked overnight at 4°C in 5% milk prepared in 1× Tris-Buffered Saline (TBS) − 0.1% Tween20 supplemented with dibenzocyclooctyne-PEG4-biotin conjugate (Sigma-Aldrich #760749; 50 µM final concentration). Membranes were washed three times in 1× TBS − 0.1% Tween20 for 10 min each, then incubated with Precision Protein StrepTactin-HRP Conjugate (BioRad #1610380; 1:1000 in 5% milk prepared in 1× TBS − 0.1% Tween20) for 1 hr at room temperature, then washed again. Membranes were subsequently developed using Amersham ECL Prime Western Blotting Detection Reagent (GE Healthcare #RPN2236). Images were acquired through the ChemiDoc MP Imaging System. ImageJ software (v 1.45s) was used for quantitation of AHA signal intensities.
9
SARS-CoV-2 S Protein Expression Analysis
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The expression of SARS-CoV-2 S protein in eVLP preparations was analyzed by western blotting as described previously [17] (link) using rabbit polyclonal Ab (pAb) anti-RBD of SARS-CoV-2 S (Sinobiological) followed by detection with goat anti-rabbit IgG-Fc horseradish peroxydase-conjugated (Bethyl). Alternatively, human sera from COVID-19 convalescent subjects was used as primary antibody followed by detection with goat anti-human IgG heavy and light chain HRP-conjugated (Bethyl). Precision Protein Streptactin HRP conjugate (Bio-Rad) was used as molecular weight ladder standard. Recombinant SARS-COV-2 S (S1 + S2) unmodified protein (Sinobiological) or SARS-CoV-2 stabilized prefusion S protein (NRC) were used as controls.
10
ELISA-Based CTLA-4 Antibody Blocking Assay
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ELISA was used to assess the blocking activities of anti-CTLA-4 antibodies. Ninety-six-well plates (Corning, 9018) were first incubated with human B7-1 (Acro Bio, B71-H5259) (2 µg/mL) or human B7-2 protein (Acro Bio, CD6-H5257) (2 µg/mL) at 4 °C overnight. The plates were then washed with PBST (Medicago AB, 09-9410-100) and blocked with 1% bovine serum albumin (Thermo Scientific, 37525) for 2 h at room temperature. Plates were next incubated with anti-CTLA-4 antibodies from max dose 800 nM, 1:3 diluted to eight doses for 20 min at room temperature, followed with 0.25 µg/mL biotin-CTLA-4 (Acro Bio, CT4-H82F3) for 1 h at room temperature. Finally, plates were incubated with Precision Protein StrepTactin-HRP Conjugate (Bio Rad, 1610380) for 30 min and developed using TMB substrate (Biopanda, TMB-S003). The optical density of samples was detected at 450 and 570 nm.
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