Cellular extracts were prepared in EBC buffer (50 mM tris (pH8.0)), 120 mM NaCl, 0.5% Nonidet P-40, 1 mM DTT and protease and phosphatase inhibitor tablets (ThermoFisher). Protein concentration was quantified by Pierce BCA assay (ThermoFisher) and samples were prepared by boiling in Laemmli buffer for 5 minutes. Equal amounts of whole cell lysates were resolved by hand-cast SDS-PAGE, and transferred to PVDF membranes (Millipore). All blocking and primary antibody steps were performed in 5% BSA diluted in TBST (137 mM NaCl, 2.7 mM KCl, 25 mM tris pH 7.4, 1% Tween-20). All primary antibody incubations were performed with shaking at 4 °C overnight. After transfer of the separated proteins to PVDF membranes, the membranes were probed with the following primary antibodies: USP37 (at dilution 1 : 1000; from Elabscience) and β-actin (at 1 : 10 000; from Sigma, MO, USA). After overnight incubation, the membranes were extensively washed, and then were incubated with either secondary goat anti-mouse/rabbit HRP-conjugated antibodies purchased from Cell Signalling Technology. Separated protein bands were visualized using a chemiluminescence HRP substrate (BioRad, CA, USA), and the membranes were imaged and analysed using the ChemiDoc™ imaging system from Bio-Rad (CA, USA).
Chemiluminescence hrp substrate
Manufactured by Bio-Rad
Sourced in United States
The Chemiluminescence HRP substrate is a laboratory equipment product designed for the detection and quantification of horseradish peroxidase (HRP) in Western blotting and other applications. It provides a sensitive and reliable method for the visualization of HRP-labeled targets.
Automatically generated - may contain errors
Lab products found in correlation
Chemidoc imaging system, by Bio-Rad (3 mentions)
Pvdf membranes, by Elabscience (2 mentions)
Usp37, by Elabscience (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Pierce bca assay, by Thermo Fisher Scientific (2 mentions)
β actin, by Merck Group (2 mentions)
Protease and phosphatase inhibitor tablet, by Thermo Fisher Scientific (1 mentions)
Mab8929, by R&D Systems (1 mentions)
Mab3925, by R&D Systems (1 mentions)
Goat anti mouse igg sc 2039, by Santa Cruz Biotechnology (1 mentions)
Sc 390991, by Santa Cruz Biotechnology (1 mentions)
γh2ax, by Santa Cruz Biotechnology (1 mentions)
Nitrotyrosine, by Merck Group (1 mentions)
Bca analysis, by Beyotime (1 mentions)
Il 1r1, by Santa Cruz Biotechnology (1 mentions)
Ripa lysis buffer, by Solarbio (1 mentions)
Bcl2 associated x (bax), by Proteintech (1 mentions)
4 protocols using chemiluminescence hrp substrate
1
Western Blot Analysis of USP37
2
Hippocampal Protein Extraction and Blotting
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Protein extraction from the hippocampal tissue and blotting analyses were performed as previously illustrated.40 (link) Nuclear factor erythroid 2-related factor 2 (Nrf2) (MAB3925, 1:1000; R&D System), heme oxygenase-1 (HO-1) (sc-390991, 1:1000; Santa Cruz Biotechnology, Santa Cruz, CA, USA), and β-actin (MAB8929, 1:2000; R&D System, Minneapolis, MN, USA) were used as a primary antibody, whereas goat anti-mouse IgG (sc-2039, 1:5000; Santa Cruz Biotechnology, Santa Cruz, CA, USA) conjugated with horseradish peroxidase (HRP) was used a secondary antibody. The protein bands were visualized using a chemiluminescence HRP substrate (Bio-Rad, Hercules, CA, USA). The blot intensity was then quantified using Image J software referenced to β-actin.
3
Western Blot Analysis of USP37 and γ-H2AX
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Cellular extracts were generated in EBC lysis buffer (50 mM Tris (pH8.0), 120 mM NaCl, 0.5% Nonidet P-40, 1 mM DTT, and protease and phosphatase inhibitor tablets) (Thermo Fisher USA). Protein concentration was quantified by Pierce BCA assay (Thermo Fisher, USA), and samples were prepared by boiling them in Laemmli buffer for 5 min. Equal amounts of whole cell lysates were resolved by hand-cast SDS-PAGE and transferred to PVDF membranes (Millipore). All blocking and primary antibody steps were performed in 5% BSA diluted in TBST (137 mM NaCl, 2.7 mM KCl, 25 mM Tris pH 7.4, 1% Tween-20). All primary antibody incubations were performed with shaking at 4℃ overnight. After the transfer of the separated proteins to PVDF membranes, the membranes were probed with the following primary antibodies: USP37 (dilution 1:1000; Elabsciences), β -actin (dilution 1:10,000; Sigma, MO, USA), γ-H2AX (at dilution 1:1000; from Santa Cruz). After overnight incubation, membranes were extensively washed and then incubated with either secondary goat anti-mouse/rabbit HRP-conjugated antibodies (Cell signalling technology). Separated protein bands were visualized using chemiluminescence HRP substrate (Bio-Rad, CA, USA), and the membranes were imaged and analyzed using the Chemi Doc™ imaging system from Bio-Rad (CA, USA).
4
Western Blot Analysis of Inflammatory Signaling
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The mouse hearts or NRVMs were lysed using RIPA lysis buffer (Solarbio, R0020) with a protease-inhibitor cocktail (Beyotime, P1030). After normalizing the total protein concentrations via the BCA analysis (Beyotime, P0010), the lysates were boiled in loading buffer for 10 min. Afterward, 20 µg lysate per sample was resolved in SDS-polyacrylamide gels (FD341; Hangzhou Fude Biological Technology Co., Ltd. Hangzhou, China), followed by transfer onto PVDF membranes for western blotting. After blocking with 5% skimmed milk for 1 h, the membranes were incubated with antibodies against IL-1R1 (1:1000, Santa Cruz, Biotechnology, Santa Clara, CA, USA), IL-1R2 (1:1000, Novus Biologicals, Littleton, CO, USA), p-NF-κB (1:1000, CST), JAK2 (1:1000, CST), p-JAK2 (1:1000, CST), STAT1 (1:1000, CST), p-STAT1 (1:1000, CST), STAT3 (1:1000, CST), p-STAT3 (1:1000, CST), Bcl-xL (1:1000, CST), Bax (1:1000, Proteintech, Rosemont, IL, USA), IL-17RA (1:1000, Abclonal, Wuhan, China), nitrotyrosine (1:1000, Sigma-Aldrich) and GAPDH (1:5000, CST) overnight at 4 °C. Afterward, the membranes were washed with TBST (2% Tween) and incubated with HRP-conjugated secondary antibodies for 1 h at room temperature. A chemiluminescence HRP substrate (Bio-Rad) and ChemiDoc Imaging System (Bio-Rad) were used to detect the protein bands.
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