Crude extracts were obtained from N. benthamiana , P. persica, and A. thaliana by homogenization of ground frozen leaf tissues in disruption buffer (50 mM Tris-HCl, pH 7.5, 6 M urea, 2% SDS, and 5% β-mercaptoethanol). After centrifugation at 13,000 × g for 10 min, samples were boiled for 5 min at 95°C. Proteins were separated in SDS-PAGE gels (12% acrylamide) and electroblotted onto a nitrocellulose membrane. The CP from PPV was detected with a specific rabbit serum used as the primary antibody and horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch) as the secondary reagent. MYC-tagged proteins were detected with a specific anti-MYC monoclonal antibody (AbMART) and HRP-conjugated sheep anti-mouse IgG (Sigma) as the secondary antibody. Immunostained proteins were visualized by enhanced chemiluminescence detection with Clarity ECL Western blotting substrate (Bio-Rad) in a ChemiDoc system (Bio-Rad). Band intensity in arbitrary units was estimated by using Image Lab software (v.6.0.0), with the signal of one selected sample considered as the reference. Ponceau red staining of membranes was used to check the global protein content of samples.
Hrp conjugated sheep anti mouse igg
Manufactured by Merck Group
Sourced in United States
HRP-conjugated sheep anti-mouse IgG is a secondary antibody that binds to mouse immunoglobulin G (IgG) molecules. The antibody is conjugated with horseradish peroxidase (HRP), an enzyme that can be used for signal amplification in various immunoassays and detection methods.
Automatically generated - may contain errors
Lab products found in correlation
Horseradish peroxidase hrp conjugated goat anti rabbit igg, by Jackson ImmunoResearch (1 mentions)
Clarity western ecl blotting substrate, by Bio-Rad (1 mentions)
Chemidoc system, by Bio-Rad (1 mentions)
Hrp conjugated goat anti rabbit igg, by Cell Signaling Technology (1 mentions)
Phospho paxillin tyr118, by Cell Signaling Technology (1 mentions)
Parp p85, by Promega (1 mentions)
Chemidoc xrs apparatus, by Bio-Rad (1 mentions)
Phospho fak tyr397, by Cell Signaling Technology (1 mentions)
Total fak, by Santa Cruz Biotechnology (1 mentions)
Total paxillin, by BD (1 mentions)
Amersham ecl, by GE Healthcare (1 mentions)
β actin, by Merck Group (1 mentions)
Quantity one software, by Bio-Rad (1 mentions)
Protein estimation kit, by Bio-Rad (1 mentions)
Mouse anti human cd3ζ, by BD (1 mentions)
Hybond nitrocellulose membrane, by Cytiva (1 mentions)
Ecl western blotting detection kit, by Cytiva (1 mentions)
Complete protease inhibitor cocktail tablet, by Roche (1 mentions)
Hrp conjugated goat anti rabbit igg, by Merck Group (1 mentions)
Anti bcl 2, by Merck Group (1 mentions)
5 protocols using hrp conjugated sheep anti mouse igg
1
Protein Extraction and Detection from Plant Tissues
2
Western Blot Analysis of SCLC Cell Lines
3
Western Blot Analysis of CD3ζ Protein
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Cells were lysed in 1% CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulphonate) buffer [phosphate-buffered saline (PBS), 1% CHAPS, 0·5% sodium deoxycholate, 2% sodium dodecyl sulphate] containing complete protease inhibitor cocktail tablets (Roche, Indianapolis, IN, USA) for 30 min, then centrifuged through polymer wool to clear the supernatant. Protein concentrations were established using a protein estimation kit (Bio-Rad, Hercules, CA, USA). Reduced or non-reduced samples containing 20 μg of total protein were separated on 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) gels, transferred to Hybond nitrocellulose membranes (Amersham Biosciences, Pittsburgh, PA, USA) and probed with mouse anti-human CD3ζ (BD Biosciences) followed by HRP-conjugated sheep anti-mouse IgG (Sigma-Aldrich), both diluted 1:1000 in PBS-Tween/5% skimmed milk powder. Bands were resolved using an ECL Western Blotting Detection kit (Amersham Biosciences), according to the manufacturer's instructions, and the membranes were analysed by exposure to X-ray film.
4
Intranasal and Intramuscular Delivery of AdC7 Vectors
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AdC7αRSV or AdC7GFP (5 × 1010 pu each) diluted in 40 μl PBS were administered intranasally or intramuscularly to 8-week-old female BALB/c mice. Neonatal mice received either 3 × 1010 pu (5μL) or 6 × 1010 pu (10 μL) of the vectors intranasally. The levels and kinetics of anti-RSV IgG following administration of AdC7αRSV were quantified in serum and bronchoalveolar lavage (BAL) by ELISA. Serial dilutions of serum and BAL were added to flat-bottomed 96-well EIA/RIA plates (Corning, Corning, NY, USA) coated with 1μg/mL of human anti-palivizumab clone AbD23967 (HCA261, Bio-Rad-Antibodies, Hercules, CA, USA), followed by PBST + 5% blotting grade blocker (Bio-Rad Laboratories, Hercules, CA, USA. Detection was performed using an HRP-conjugated sheep anti-mouse IgG (Sigma, St. Louis, MO, USA) in PBS + 1% blotting grade blocker and substrate (hydrogen peroxide/tetramethylbenzidine) (R&D systems, Minneapolis, MN, USA) and the absorbance at 450 nm was measured. Titers were calculated with a log (OD)–log(dilution) interpolation model, with detection cut-off equal to 2-fold the background absorbance. Half-life (t1/2) was calculated by the formula: where t = time elapsed, N0 = titer at 1 week, and Nt = titer at 4 weeks after the administration of AdC7αRSV.
5
Protein Lysis and Western Blot Analysis
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Cells were lysed in ice-cold lysis buffer (20 mm Tris-HCl pH8, 300 mm KCl, 10% Glycerol, 0.25% Nonidet P-40, 0.5 mm EDTA, 0.5 mm EGTA, 1 × protease inhibitors) and lysates cleared by centrifugation (25 min, 15 000 × g, 4 °C) before PAGE and western blotting as previously described.10 (link) Primary antibodies used for western blot analysis were used at the dilution recommended by the manufacturer unless noted, and included a custom PRP4K antibody (H143, 1:1000);9 (link) antibodies from Cell Signaling (Danvers, MA, USA), anti-EGFR (#4267, 1:2000), anti-Akt (pan) (#4691, 1:2000), anti-p-Akt (Ser473) (#4060, 1:2000), anti-Erk (#4695), anti-pErk (#9106), anti-Bcl2 (#3498) and antibodies in the EMT sampler kit (#9782); Sigma, anti-actin (A2228) (1:5000); and Santa Cruz (Santa Cruz, CA, USA), anti-TrkB (sc-37721). Secondary antibodies used include HRP-conjugated goat anti-rabbit IgG (Sigma, A6154), HRP-conjugated sheep anti-mouse IgG (Sigma, A5906) and HRP-conjugated donkey anti-sheep IgG (Sigma, A3415).
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