At 48h after transfection, cells were rinsed twice with PBS and then harvested in PBS containing protease inhibitors (Complete tablet from Roche). The cells were lysed in PBS, 1% Igepal and protease inhibitors for 30min on ice. The detergent lysates were then clarified by centrifugation (14,000×g, 30min, 4°C). Proteins were separated by SDS-PAGE on 3-8% Tris-Acetate or 4-12% Bis-Tris gels and then transferred to polyvinylidene fluoride membranes. After blocking in TBS buffer (10mM Tris, pH 7.4, 500mM NaCl. 0.5% Igepal, 10% goat serum and 3% BSA), the membranes were incubated with primary antibody overnight. The protein-Ab complexes were then labeled with a horseradish peroxidase-conjugated secondary Ab (1:3000, Sigma-Aldrich) for 1h at room temperature and detected using the enhanced ECL Plus reagent (GE Healthcare) visulalized with a Typhoon 9410 scanner (GE Healthcare). Quantification of immunoblot bands was performed with ImageQuant software (GE Healthcare). The following Abs were used: rabbit anti-CaV2.2 (1:500)53 (link), rabbit anti-CaVβ1b (1:500)22 (link) and mouse anti-CaVα2δ-1 (1:3000, D219, Sigma).
Horseradish peroxidase conjugated secondary ab
Manufactured by Merck Group
Sourced in United States
Horseradish peroxidase-conjugated secondary Ab is a laboratory reagent used in various immunoassay techniques. It consists of a secondary antibody that is conjugated to the enzyme horseradish peroxidase. This enzyme can catalyze a colorimetric or chemiluminescent reaction, allowing for the detection and quantification of target analytes in a sample.
Automatically generated - may contain errors
Lab products found in correlation
Typhoon 9410 scanner, by GE Healthcare (3 mentions)
Complete tablet, by Roche (3 mentions)
Ecl plus reagent, by GE Healthcare (2 mentions)
Imagequant software, by GE Healthcare (2 mentions)
Nisin, by MoBiTec (1 mentions)
Sars cov 2 spike protein, by GenScript (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Fujifilm (1 mentions)
Mouse anti β actin mab ac 15, by Merck Group (1 mentions)
7 protocols using horseradish peroxidase conjugated secondary ab
1
Western Blot Analysis of Cav Subunits
2
Western Blot Protein Analysis Protocol
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Forty-height hours after transfection, cells were rinsed twice with PBS and then harvested in PBS containing protease inhibitors (cOmplete tablet, Roche). The cells were lysed in PBS containing 1% Igepal and protease inhibitors for 30 min on ice. The detergent lysates were then clarified by centrifugation (14,000 ×g, 30 min, 4 °C). Proteins were separated by SDS-PAGE on 3–8% Tris-Acetate or 4–12% Bis-Tris gels and then transferred to polyvinylidene fluoride membranes. After blocking in TBS buffer (10 mM Tris, pH 7.4, 500 mM NaCl. 0.5% Igepal, 10% goat serum and 3% BSA), the membranes were incubated with primary antibody overnight. The protein-Ab complexes were then labelled with a horseradish peroxidase-conjugated secondary Ab (Sigma-Aldrich) for 1 h at room temperature and detected using the enhanced ECL Plus reagent (GE Healthcare) visualized with a Typhoon 9410 scanner (GE Healthcare). Quantification of immunoblot bands was performed with ImageQuant software (GE Healthcare) or Image J.
3
Western Blot Analysis of Calcium Channel Subunits
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At 48 h after transfection, cells were rinsed twice with
phosphate-buffered saline (PBS) and then harvested in PBS containing protease
inhibitors (Complete tablet from Roche). The cells were lysed in PBS, 1% Igepal
and protease inhibitors for 30 min on ice. The detergent lysates were
then clarified by centrifugation (14,000 g, 30 min,
4 °C). Proteins were separated by SDS–PAGE on
3–8% Tris-acetate
or 4–12% Bis-Tris gels and then transferred to polyvinylidene
fluoride membranes. After blocking in Tris-buffered saline buffer
(10 mM Tris, pH
7.4, 500 mM NaCl.
0.5% Igepal, 10% goat serum and 3% BSA), the membranes were incubated with
primary antibody overnight. The protein–Ab complexes were then
labelled with a horseradish peroxidase-conjugated secondary Ab (1:3,000
Sigma-Aldrich) for 1 h at room temperature and detected using the
enhanced ECL Plus reagent (GE Healthcare) visualized with a Typhoon 9410 scanner
(GE Healthcare). Quantification of immunoblot bands was performed with
ImageQuant software (GE Healthcare). The following Abs were used: rabbit
anti-CaV2.2
(1:500)52 (link), rabbit anti-CaVβ1b (1:500) and
mouse anti-CaVα2δ-1
(1:3,000, D219, Sigma).
phosphate-buffered saline (PBS) and then harvested in PBS containing protease
inhibitors (Complete tablet from Roche). The cells were lysed in PBS, 1% Igepal
and protease inhibitors for 30 min on ice. The detergent lysates were
then clarified by centrifugation (14,000 g, 30 min,
4 °C). Proteins were separated by SDS–PAGE on
3–8% Tris-acetate
or 4–12% Bis-Tris gels and then transferred to polyvinylidene
fluoride membranes. After blocking in Tris-buffered saline buffer
(10 mM Tris, pH
7.4, 500 mM NaCl.
0.5% Igepal, 10% goat serum and 3% BSA), the membranes were incubated with
primary antibody overnight. The protein–Ab complexes were then
labelled with a horseradish peroxidase-conjugated secondary Ab (1:3,000
Sigma-Aldrich) for 1 h at room temperature and detected using the
enhanced ECL Plus reagent (GE Healthcare) visualized with a Typhoon 9410 scanner
(GE Healthcare). Quantification of immunoblot bands was performed with
ImageQuant software (GE Healthcare). The following Abs were used: rabbit
anti-CaV2.2
(1:500)52 (link), rabbit anti-CaVβ1b (1:500) and
mouse anti-CaVα2δ-1
(1:3,000, D219, Sigma).
4
Optimizing SARS-CoV-2 Spike Protein Expression
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HCR spike gene expression was induced by supplementation of various nisin concentrations (MoBiTec GmbH, Goettingen Germany) to determine the optimal condition for protein expression. Various nisin concentrations (0–80 ng/mL) were added to the culture medium at the OD600 of around 0.8 followed by incubation at for 18 h at 30 °C. Bacterial cells and culture supernatants were separated by centrifugation, and the cells were sonicated five times. The expression of the intracellular and extracellular proteins was analyzed using SDS-PAGE. Furthermore, Western blotting was performed using a primary antibody (Ab) against the SARS-CoV-2 spike protein (0.5 ug/mL, GenScript, New Jersey, USA), followed by incubation with a horseradish peroxidase-conjugated secondary Ab (1:100.000; Sigma, St. Louis, MO, USA) for detection of the intracellular spike protein.
5
Mouse Organ Protein Analysis
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Mouse organs (the heart, muscle, kidney, liver, and epididymis) were collected from 8–12-week-old ICR male mice and the sperm were collected from the epididymis. The tissues were lysed by placing in Laemmli’s sodium dodecyl sulfate (SDS) sample buffer [2% (w/v) SDS, 62.5 mM Tris-HCl (pH 6.8), 0.005% bromophenol blue, and 7% glycerol] and boiling for 10 min at 95 °C. The protein samples were resolved by SDS polyacrylamide gel electrophoresis on 12% acrylamide gel, and immunoblotted. Detection of the proteins and the relevant primary Abs (0.1 μg/ml) was based on enzyme-linked color development with horseradish peroxidase-conjugated secondary Abs (0.01 μg/ml; Sigma-Aldrich).
6
Immunoblotting of Neuronal Nuclei Protein
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Cultured undifferentiated and differentiated SK-N-SH cells on 100 mm culture dishes were lysed using RIPA lysis buffer. The lysed supernatants were denatured by adding Laemmli's sodium dodecyl sulfate (SDS) sample buffer (2% [w/v] SDS, 62.5 mM Tris-HCl [pH 6.8], 0.005% bromophenol blue, and 7% glycerol) and heated at 95 °C for 10 min. The samples (5 μg/20 μL) were subjected to SDS polyacrylamide gel electrophoresis on 12% acrylamide gels prior to immunoblotting. Immune complexes of anti-NeuN polyclonal Abs (0.1 μg/mL) were determined using enzyme-linked color development with horseradish peroxidase-conjugated secondary Abs (0.1 μg/mL; 1:10,000, Sigma–Aldrich).
7
Immunoblotting and Immunocytochemistry of Mouse eCS
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For immunoblotting and immunocytochemistry experiments, a rabbit polyclonal antibody (polyAb) against a synthetic peptide corresponding to the N-terminus (IYRNLYREDRNIEA) of the mouse eCS (GenBank accession no. NP_082221.2, 1/250 dilution for immunoblotting, 1/100 dilution for immunostaining) was produced by Japan Lamb Ltd (Hiroshima, Japan). Rabbit anti-CS polyAb (NE040/7S, 1/500 dilution for immunoblotting, 1/100 dilution for immunostaining) and anti-PLCz1 polyAb (ab181816, 1/250 dilution for immunoblotting, 1/50 dilution for immunostaining) were purchased from Nordic MUbio (Susteren, Netherlands) and Abcam Inc. (Cambridge, MA), respectively. Mouse anti-β-actin mAb (AC-15, 1/500 dilution for immunoblotting) and anti-glyceraldehyde-3-phosphate dehydrogenase mAb (5A12, 1/500 dilution for immunoblotting) were purchased from Sigma-Aldrich (St. Louis, MO) and WAKO Pure Chemical Industries (Osaka, Japan). IgGs conjugated with Alexa Fluor 488 and 546 were purchased from Molecular Probes (Invitrogen, Carlsbad, CA) as secondary antibodies for immunohistochemistry. Horseradish peroxidase-conjugated secondary Abs (Sigma-Aldrich) were used for immunoblotting. The nuclei were counterstained with 4′,6-diamidino-2-phenylindole (WAKO Pure Chemical Industries, Osaka, Japan).
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