The 20 or 50 μg cell lysates were subjected to SDS-PAGE or Phos-tag SDS-PAGE. The SDS-polyacrylamide gels consisted of an 8% or 10% acrylamide separation gel and a 3% stacking gel. The Phos-tag gels consisted of a separating gel copolymerized with Phos-tag (6% acrylamide, 20 μM acrylamide-pendant Phos-tag, 40 μM MnCl2). After electrophoresis, Phos-tag gels were soaked in a transfer buffer (25 mM Tris, 192 mM glycine, and 20% methanol) containing 1 mM EDTA for 20 min with gentle agitation for elimination of the manganese ions from the gel. Next, the gels were soaked in a transfer buffer without EDTA for 10 min with gentle agitation. The resolved proteins were electrophoretically transferred to nitrocellulose membranes (GE Healthcare Biosciences, Protran BA85). The membranes were incubated with Multi-PK antibody (YK34), anti-phospho-PKA antibody, anti-Syk antibody diluted 1:200 or an anti-PKA antibody, anti-Erk1/2 antibody, anti-phospho-Erk1/2 antibody diluted 1:1000 for 2 h at room temperature. The membranes were then incubated with horseradish peroxidase conjugated anti-mouse IgG (Sigma) diluted 1:2000 or horseradish peroxidase conjugated anti-rabbit IgG (Cappel) diluted 1:5000 for 1 h, and the immunoreactive bands were detected using the chemiluminescent substrate, Western Lightning Plus-ECL (PerkinElmer).
Horseradish peroxidase conjugated anti mouse igg
Manufactured by Merck Group
Sourced in United States, United Kingdom
Horseradish peroxidase-conjugated anti-mouse IgG is a laboratory reagent used to detect and quantify mouse immunoglobulin G (IgG) in various immunological assays. The horseradish peroxidase (HRP) enzyme is conjugated to the anti-mouse IgG antibody, allowing for the visualization and measurement of mouse IgG in samples.
Automatically generated - may contain errors
Lab products found in correlation
Western lightning plus ecl, by PerkinElmer (2 mentions)
Enhanced chemiluminescence detection system, by Thermo Fisher Scientific (2 mentions)
Nupage 4 12 bis tris gel, by Thermo Fisher Scientific (2 mentions)
Primary anti sod 1, by Santa Cruz Biotechnology (2 mentions)
Image lab software, by Bio-Rad (2 mentions)
Chemidoc mp, by Bio-Rad (2 mentions)
Multiskan ascent microplate reader, by Thermo Fisher Scientific (2 mentions)
Alkali soluble casein, by Merck Group (2 mentions)
Protran ba85, by GE Healthcare (1 mentions)
Hybond enhanced chemiluminescence ecl nitrocellulose membrane, by Cytiva (1 mentions)
Anti gfp monoclonal antibody, by Takara Bio (1 mentions)
Chemidoc xrs system, by Bio-Rad (1 mentions)
Elisa plate reader, by Thermo Fisher Scientific (1 mentions)
Igg2c, by Southern Biotech (1 mentions)
Igg2b, by Southern Biotech (1 mentions)
3 3 5 5 tetramethylbenzidine substrate, by Merck Group (1 mentions)
Ecl reagent, by GE Healthcare (1 mentions)
Bis tris gel, by Thermo Fisher Scientific (1 mentions)
Ecl imager, by Bio-Rad (1 mentions)
Protease phosphatase inhibitor, by Merck Group (1 mentions)
26 protocols using horseradish peroxidase conjugated anti mouse igg
1
SDS-PAGE and Phos-tag Immunoblotting Analysis
2
MCMV IE1 Protein Detection in MEF Cells
3
Western Blot Analysis of GFP
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Equal amounts of TCP (10 µg) were mixed with SDS-PAGE loading buffer and boiled at 100 °C for 3 min. 10% SDS-polyacrylamide gels was used to separate the protein samples. After electrophoresis, proteins were blotted onto a Hybond enhanced chemiluminescence (ECL) nitrocellulose membrane (Amersham, Arlington Heights, IL, USA) using the wet transblotting method. The primary antibody was anti-GFP monoclonal antibody (Clontech) and the secondary antibody was a horseradish peroxidase-conjugated anti-mouse IgG (Sigma, St Louis, MO, USA). When used, their dilution ratios were 1:1000 and 1:5000 dilution, respectively. The Western blots were detected by using standard ECL reagents (Pierce, Rockford, IL, USA) and imaged by using a ChemiDoc XRS system (Bio-Rad, Hercules, CA, USA).
4
Immunoblotting Analysis of Cytochrome P450 Enzymes
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Whole cell lysates from cells (human embryonic kidney cells-HEK 293) overexpressing CYP2R1, CYP7B1, CYP8B1, CYP27A1, CYP39A1, CYP46A1 and CYP51A1 respectively were used as positive controls for immunoblotting while lysates from cells containing vector only were used as negative controls. The cell lysates and their corresponding controls were obtained from (Novus Biologicals, Cambridge, UK). Cell lysates (5 μg protein/lane) were resolved by electrophoresis on NuPAGE 4-12% Bis-Tris gels (Fisher Scientific, Loughborough, UK). Following protein transfer to nitrocellulose membrane the membranes were washed for 45 minutes at room temperature in phosphate buffered saline-Tween-20 (PBST) containing 3 % (w/v) skimmed milk powder to block non-specific protein binding. Membranes were incubated overnight at 4°C with individual monoclonal antibodies diluted in PBST (1/2 dilution) and then washed 6 times for a total of 60 minutes in 1% skimmed milk. The membranes were subsequently probed for 60 minutes with a secondary antibody conjugated horseradish-peroxidase-conjugated anti-mouse IgG (1/2000, Sigma-Aldrich, Dorset, UK). Membranes were then washed (6 times) for a total of 60 minutes in 1% skimmed milk and protein bands visualized using the enhanced chemiluminescence detection system (Fisher Scientific) [26 (link), 49 (link)].
5
Quantitative ELISA for Anti-BSA Antibody
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Anti-BSA antibody levels were determined with ELISA. For this purpose, polystyrene 96-well plates were coated overnight at 4 °C with BSA (500 ng per well) diluted in 0.2 M carbonate buffer (pH 9.6). Each incubation step was preceded by three washes with PBST (PBS 1× + 0.05% Tween 20). The plates were blocked with a fat-free powder milk solution (5%) at room temperature for 2 h. Serial dilutions of the test sera were applied and incubated overnight at 4 °C. Horseradish peroxidase-conjugated anti-mouse IgG (1:2000 dilution; Sigma-Aldrich, St. Louis, MO, USA), IgG1, or IgG2a were used for secondary labeling (2 h of incubation at 25 °C). The reaction was developed by adding a substrate solution of 0.3 mg/L ABTS and 0.1 M H2O2 followed by 50 min of incubation at 25 °C. OD405nm values were measured in a Multiskan Ascent microplate reader (Thermo Fisher Scientific). The statistical significance (p-value) was determined using one-way ANOVA. The statistical analysis was performed using Statistica® 12.7 (TIBCO Software Inc., Palo Alto, CA, USA).
6
SARS-CoV-2 Antigen-Specific IgG ELISA
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The antigen-specific IgG antibody responses were detected by ELISA as described previously [22] (link). Briefly, serially diluted mouse sera were added to 96-well micro-well plates pre-coated with rNTD protein at 100 ng per well. After blocking with FBS and washing with PBST, bound antibodies were reacted with horseradish peroxidase-conjugated anti-mouse IgG (1:5000, Sigma-Aldrich, St. Louis, MO, US) at 37 °C for 1 h. The samples were then washed with PBST, after which 3,3′,5,5′-tetramethylbenzidine was added to the plates. The reaction was stopped by adding 2 M H2SO4. The absorbance was read at 450 nm using an ELISA plate reader (Thermo, Waltham, MA,US). The cut-off value was set to 2.1-fold above that of the negative control.
7
SARS-CoV-2 Antibody Isotyping ELISA
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High‐binding 96‐well plates (Nunc, Thermo Fisher Scientific, MA, USA) were coated with the antigen (S1 protein 1.2 μg mL−1; S2 0.8 μg mL−1; SCV 105 plaque‐forming units/well) in carbonate buffer (pH 9.8) and incubated at 4°C overnight. Plates were washed, blocked and incubated with serial dilutions of serum in 1% skim milk powder for 2 h at room temperature. Binding antibodies were detected using horseradish peroxidase‐conjugated anti‐mouse IgG (catalog number A9044; Sigma Aldrich, St. Louis, MO, USA), IgG1 (catalog number 1070‐05; Southern Biotech, AL, USA), IgG2b (catalog number 1090‐05; Southern Biotech, AL, USA), IgG2c (catalog number 1079‐05; Southern Biotech, AL, USA), IgG3 (catalog number 11100‐05; Southern Biotech, AL, USA) and signals developed using 3,3′,5,5′‐tetramethylbenzidine substrate (catalog number T0440; Sigma‐Aldrich, St. Louis, MO, USA). Endpoint titers were defined as the highest reciprocal of serum dilution to yield an absorbance equal to the negative serum samples plus three times the standard deviation.
8
Antibody Response and Neutralization Assay
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Antigen-specific IgG antibodies in mouse sera were measured by indirect ELIZA. Briefly, 96-well ELIZA plates (Nunc, USA) were coated overnight at 4 °C with 50 ng/well of EV71-VLP, CVA16-VLP, CVA10-VLP, or CVA6-VLP, followed by blocking in 5% milk in PBS-Tween20 (PBST). Serum samples collected two weeks after the last immunizations were added at a dilution of 1:1000 (50 μL/well) and incubated for 2 h at 37 °C. The plates were then incubated with horseradish peroxidase-conjugated anti-mouse IgG (Sigma-Aldrich, USA). Plates were washed three times with PBST between each step. TMB substrate (New Cell & Molecular Biotech, China) was added for color development, and then the absorbance at 450 nm was determined.
The neutralization titers of antisera against EV71, CVA16, CVA10, and CVA6 were determined by micro-neutralization assays as described previously28 (link),29 (link),37 (link),40 (link). Neutralization titers are defined as the highest serum dilutions at which no cytopathic effects are observed.
The neutralization titers of antisera against EV71, CVA16, CVA10, and CVA6 were determined by micro-neutralization assays as described previously28 (link),29 (link),37 (link),40 (link). Neutralization titers are defined as the highest serum dilutions at which no cytopathic effects are observed.
9
Quantifying Amyloid-Beta Aggregation
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npAβ and pS8Aβ (50 μM, pH 7.4 buffered with 20 mM sodium phosphate containing 50 mM sodium chloride) were incubated at 37 °C with gentle stirring. Aliquots of samples collected at different time points were centrifuged at 100,000g for 30 min to separate the soluble Aβ species in the supernatant from insoluble Aβ assemblies in the pellet. The supernatant and pellets were subjected to native-PAGE or denaturing SDS–PAGE and Aβ was detected by western blotting. The samples were mixed with an equal volume of 4 × SDS sample buffer and then boiled at 100 °C for 5 min. The samples were electrophoresed on 4–12% Bis-Tris gels (Novex, San Diego, CA, USA) and then transferred onto 0.2-μM nitrocellulose membrane (Whatmann GmbH, Germany) at 400 mA for 2 h at 4 °C. After 2 h, membranes were washed in TBS-T and incubated overnight with the primary antibody 82E1 in 0.5 μg ml−1 TBS-T solution at 4 °C. The bound antibody was detected using horseradish peroxidase-conjugated anti-mouse IgG (1:50,000; Sigma) using enhanced chemiluminescence (ECL reagent, GE Healthcare) with an ECL imager (BioRad Inc.).
10
Western Blot Analysis of Cell Cycle Regulators
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Protein samples were prepared in ice-cold radioimmunoprecipitation assay buffer supplemented with protease/phosphatase inhibitors (Sigma Aldrich; Merck KGaA) for 30 min. Lysates were cleared by centrifugation at 10,000 × g for 10 min at 4°C. Protein samples (50 µg in 10 µl loading buffer per lane) were resolved by 12% SDS-PAGE and transferred to nitrocellulose membranes. Membranes were blocked with 5% fat-free milk at 37°C for 1 h and incubated with primary antibodies at 4°C overnight. The primary antibodies used are summarized as follows: Against cyclin D1 (sc-70899; 1:500 dilution), cyclin-dependent kinase 2 (CDK2) (sc-6248; 1:500 dilution), S-phase kinase-associated protein (SKP)2 (sc-74477; 1:800 dilution), p21 (sc-6246; 1:1,000 dilution), p27 (sc-53906; 1:1,000 dilution), and β-actin (sc-47778; 1:2,000 dilution) (all Santa Cruz Biotechnology, Inc., Dallas, TX, USA). The membranes were then incubated with horseradish peroxidase-conjugated anti-mouse IgG (1:5,000 dilution; A9044; Sigma-Aldrich; Merck KGaA) at room temperature for 1 h. Immunoreactive signals were visualized by chemifluorescence using ECL Plus Western Blotting Detection reagents (GE Healthcare Bio-Sciences, Pittsburgh, PA, USA). Protein signals were quantified using Quantity One software (version 4.6.2; Bio-Rad Laboratories, Hercules, CA, USA). Each assay was repeated three times.
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