Antibody production was quantified from the peripheral blood of mice obtained at 0, 15, 30, and 45 days after immunization using the indirect ELISA method [25 (link)]. For this, 96-well plates (Nunc Maxisorp, Thermo Fisher Scientific, MA, USA) were coated with 5 μg of recombinant proteins per milliliter of carbonate-bicarbonate buffer (50 mM, pH 9.6) and incubated for 16 h at 4°C in a humidity chamber. Plates were washed with TPBS buffer (PBS plus 0.05% Tween 20) and blocked with 0.8% gelatin for 1 h at 37°C in order to avoid nonspecific binding. To each well, 100 μl of serum was added in serial dilutions using TPBS plus 0.2% gelatin, starting at 1 : 200 dilutions. Samples were incubated for 3 h at 37°C. After this time, rabbit anti-mouse IgM, IgG, IgG1, or IgG2a secondary antibodies conjugated with horseradish peroxidase (Serotec, Oxford, UK) diluted 1 : 1000 were added and incubated for 45 min at 37°C. The reaction was revealed with 100 μl SigmaFast (Sigma-Aldrich, St. Louis, MO, USA) OPD. The final reaction was stopped with 50 μl of sulfuric acid 2 N and read at 490 nm using a VictorX3 microplate reader (PerkinElmer, USA). Results were expressed as mean ± standard deviation (SD) of the inverse from the last dilution reached before the cut-off. All experiments were done in triplicate.
Horseradish peroxidase
Manufactured by Bio-Rad
Sourced in United States, United Kingdom
Horseradish peroxidase is an enzyme that catalyzes the oxidation of various substrates in the presence of hydrogen peroxide. It is a commonly used reporter enzyme in various biochemical and immunological assays.
Automatically generated - may contain errors
Lab products found in correlation
Nitrocellulose membrane, by Bio-Rad (7 mentions)
Complete protease inhibitor, by Roche (6 mentions)
Gapdh, by Santa Cruz Biotechnology (4 mentions)
Protease inhibitor cocktail, by Roche (4 mentions)
Enhanced chemiluminescence reagent, by Bio-Rad (4 mentions)
Nitrocellulose membrane, by Cytiva (4 mentions)
Polyvinylidene difluoride membrane, by Bio-Rad (4 mentions)
Anti β actin, by Merck Group (4 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Pierce ecl system, by Thermo Fisher Scientific (3 mentions)
Complete protease inhibitor cocktail tablet, by Roche (3 mentions)
Gel doc, by Bio-Rad (3 mentions)
Polyvinylidene fluoride membrane, by Merck Group (3 mentions)
Ecl detection kit, by Cytiva (3 mentions)
Polyvinylidene difluoride membrane, by Merck Group (3 mentions)
β actin, by Merck Group (3 mentions)
Enhanced chemiluminescence, by Biosesang (3 mentions)
Chemidoc touch imager, by Bio-Rad (3 mentions)
Bradford assay, by BioLegend (3 mentions)
Cellevent caspase 3 7 green detection reagent, by Thermo Fisher Scientific (3 mentions)
110 protocols using horseradish peroxidase
1
Indirect ELISA for Antibody Quantification
2
Enzyme-Linked Immunosorbent Assay
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Polystyrene microtiter plates (Nunc, Roskilde, Denmark) were coated (overnight at 4 °C) with histone (2.5 μg ml−1), heparin sodium salt (2 μg ml−1), carbonic anhydrase (5 μg ml−1), actin or tubulin (5 μg ml−1), thyroglobulin (10 μg ml−1), F(ab′)2 fragments (1 μg ml−1) or trinitrophenyl–bovine serum albumin (10 μg ml−1) in 0.1 M carbonate-bicarbonate buffer, pH 9.6, whereas native DNA (10 μg ml−1) was coated in PBS, pH 6.8. The antigen-coated plates were washed with PBS, blocked with PBS–BSA, and then incubated with the samples (the three IVIg, Intraglobin F-IVIg affinity-purified antibody fractions and respective effluents; 150–1.17 μg ml−1) for 2 h at 37 °C. The wells were washed with PBS containing 0.1% Tween-20 (Merck, Darmstadt, Germany; PBS–T), and sheep anti-human IgG (H- and L-chain-specific) conjugated to horseradish peroxidase (0.8 μg ml−1 in PBS–T–BSA; Serotec, Oxford, UK) was added and incubated for 90 min at 37 °C. In human IgG F(ab′)2-coated plates, goat anti-human IgG (γ-chain-specific) conjugated to alkaline phosphatase (0.5 μg ml−1 in PBS–T–BSA; Sigma) was used. The enzyme substrate, tetramethyl-benzidine (Seramun, Germany) or p-nitrophenyl-phosphate (Sigma), was added and OD was measured at 450 or 405 nm, respectively, with an ExpertPlus microplate reader (Asys, Eugendorf, Austria).
3
Western Blot Protein Detection
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The following antibodies were used as primary antibodies: mouse monoclonal anti-Flag (1:5000; Sigma), mouse monoclonal anti-6xHis (1:2500), rabbit polyclonal anti-PBP2 (1:1000; lab collection), rabbit polyclonal anti-PBP3 (1:1000, lab collection) [21] . Goat polyclonal anti-mouse (1:20,000) or anti-rabbit IgG (1:20,000) conjugated to horseradish peroxidase (Bio-Rad) were used as secondary antibodies. Proteins were separated by 4-20% precasted polyacrylamide gels, 7% tricine gels, or 6% acrylamide gels.
4
Antibody Western Blot Protocol
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The following antibodies were used as primary antibodies: mouse monoclonal anti-FLAG (clone M2; Sigma), mouse monoclonal anti-6×His (Clontech), rabbit polyclonal anti-IgaA (42 (link)), rabbit polyclonal anti-OmpA (gift from H. Schwarz, University of Tübingen, Germany), and rabbit polyclonal anti-PBP3 (our lab collection). Goat polyclonal anti-mouse or anti-rabbit IgG conjugated to horseradish peroxidase was used as a secondary antibody (Bio-Rad). SDS-PAGE and Western blotting were performed as described previously (33 (link)).
5
Immunoblot Analysis of Signaling Proteins
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The steady-state levels of ERK5, p-ERK5, MEK5, NF-κB, p-NF-κB, IκB-α, p-IκB-α, Vimentin, β-actin, HDAC and GAPDH were determined by immunoblot analysis, as previously described.44 (link) Briefly, blots were incubated overnight at 4 °C with primary rabbit antibody reactive to ERK5 (#3372), p-ERK5 (#3371) or primary mouse antibody reactive to p-IκB-α (#9246; all from Cell Signalling) or primary rabbit antibody reactive to NF-κB (#sc-372), IκB-α (#sc-371) or primary mouse antibody reactive to MEK5 (#sc-135986), Vimentin (#sc-32322), GAPDH (#sc-32233), β-actin (#sc-8432; all from Santa Cruz Biotechnology) or HDAC (#05-614, Merck Millipore Corp, Billerica, MA, USA) or primary rabbit antibody reactive to p-NF-κB (#ab131109; Abcam). Next, immunoblots were incubated with anti-rabbit or -mouse secondary antibody conjugated with horseradish peroxidase (Bio-Rad Laboratories, Hercules, CA, USA) for 3 h at RT. The relative intensifies of protein bands were analysed using the densitometric analysis software Image Lab version 5.1—beta, using a Chemidoc MP Imaging System for acquisition (Bio-Rad Laboratories).
6
Hypothalamic AMPK Activation Assay
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Anesthetized rats were decapitated, and the hypothalamus was removed and processed for Western blotting as previously described [17 (link)]. The hypothalamic tissue removed for Western blot analysis contained approximately 4.0 mm3 and had the optic chiasm as the rostral limit (bregma −0.25 mm), the infundibular stem as the caudal limit (bregma −4.20 mm) and were 4.0 mm wide and 3.0 mm deep [24 ]. The extractions occurred two hours before lights off (ZT 10). The primary antibodies used were as follows: anti-pAMPK alpha (T172) from Cell Signaling Technology (Cat. 2531S, Danvers, MA, USA) and anti-GAPDH from Cell Signaling (Cat. 2118S, Danvers, MA, USA). Secondary antibodies conjugated with horseradish peroxidase (Bio-Rad Laboratories, Hercules, CA, USA) were used, followed by chemiluminescent detection of the bands on X-ray-sensitive films. Optical densitometry was performed using the Scion Image analysis software (version, Scion Corp., Frederick, MD, USA).
7
Ginsenoside Rg3 Modulates Smooth Muscle Markers
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WPMY-1 and NAF cells were treated with vehicle or ginsenoside Rg3 for 72 h. Proteins of cell extracts were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride (PVDF) membranes. The membranes were incubated with antibodies against SM22, SMMHC, and GAPDH (KC-5G4, Kangchen, China) overnight at 2 -8 °C. Proteins were detected by appropriate secondary antibodies (goat anti-rabbit or goat anti-mouse) conjugated with horseradish peroxidase (Bio-Rad, USA) followed by enhanced chemiluminescence detection (Amersham, USA). The results were quantified with Image J software and expressed as mean of triplicates ± SD.
8
Western Blot Analysis of Protein Lysates
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Total cell lysates were prepared in RIPA buffer (50 mM Tris-Cl pH 7.4, 150 mM NaCl, 1% sodium deoxycholate, 1% NP-40; 0.1% SDS) or cell lysis buffer (20 mM Tris-Cl pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na3VO4, 1% Triton-X-100) containing protease inhibitor cocktail (Roche) and phosphatase inhibitor (Sigma) and protein concentrations were determined by BCA assay kit (Thermo Scientific, Waltham, MA). Thirty micrograms of total cell lysates were subjected to 4–20% SDS-PAGE and transferred to polyvinylidene difluoride membrane. The membrane was blocked in 20 mM Tris-HCl, 137 mM NaCl, and 0.2% Tween 20 (pH 7.6) containing 5% nonfat milk. The membrane was immunoblotted with primary antibodies at 4 °C overnight followed by secondary antibody conjugated with horseradish peroxidase (Bio-Rad, Hercules, CA) for 1 hr. The membrane was exposed on X-ray film using ECL Western blot detection reagents (Pierce Biotechnology, Rockford, IL) or digitally captured using Odyssey blot imager (LI-COR, Lincoln, NE). Raw uncropped images with molecular size marker were displayed in the supplementary section (Figs S4 –S9 ). Immunoblot band density was measured by ImageJ program (National Institute of Health) and normalized by the intensity of loading control (Tubulin or Actin) as indicated.
9
Hepatic Protein Analysis for HIF Factors
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Dissected livers were snap-frozen in liquid nitrogen and protein lysates for the detection of HIF1α and HIF2α proteins were extracted with the NE-PER kit (Pierce). Primary hepatocytes and Hep3B cells were lysed in buffer of 50 mM Тris–HCl, pH 8, 150 mM NaCl, 0.5% NP-40, 1 mM PMSF and a complete protease inhibitor cocktail tablet (Roche), kept on ice for 20 min and centrifuged at 12000 g for 20 min at 4 °C. 100 μg of proteins from liver or cells were resolved by SDS-PAGE, blotted, and probed with the following primary antibodies: Fmo5, PPARα, caspase-3, NF-kB p65, IL-6, IL-4, TNF-a, and GAPDH. The secondary antibody, either anti-mouse or anti-rabbit, was conjugated to horseradish peroxidase (1:5000; Bio-Rad Laboratories). The Pierce ECL system (ThermoScientific) was used for detection.
10
Protein Extraction and Western Blot Analysis
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Ice-cold PBS was used to rinse cells, followed by lysis with lysis buffer (50 mM Tris-HCl (pH 8.0), 120 mM NaCl, 5 mM EDTA, 0,5% Triton X-100) containing PMSF (1 mM), proteinase inhibitors (Roche, Mannheim, Germany, #1697498) and phosphatase inhibitors (10 mM NaPPi, 200 μM NaVO3, 25 mM NaF). Incubation for 15 min on ice was followed by centrifugation of lysates at 16 000 g for 20 min. Bradford Assays (Bio-Rad, Munich, Germany) were used to measure protein concentrations. For electrophoresis, 20-40 μg of protein was separated with 10-15% polyacrylamid gels and blotted onto nitrocellulose membranes (Bio-Rad) by standard procedures. Membranes where washed, incubated over night with primary antibody, washed again and incubated with secondary antibody (1:3000) coupled to horseradish peroxidase (Bio-Rad). Visualization was performed by an enhanced chemiluminescence detection system (GE Healthcare, Munich, Germany). Following primary antibodies were used: anti-beta-Actin (Sigma, Deisenhofen, Clone AC15, A5441), anti-4ebp1 (Cell Signaling, Boston, #9644), anti-p-4ebp1 (Cell Signaling, #2855), anti-rpS6 (Cell Signaling, #2317), anti-p-rpS6 (Cell Signaling, #4858), anti-p27 (DakoCytomation, Clostrup, Clone SX53G8, M 7203). Everolimus was usually used in concentrations of 1 μM for 72 h, leucine (Sigma-Aldrich, St. Louis, MO) for 2 h at concentrations of 10 mM.
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