Proteins were extracted from the cell pellet by RIPA buffer comprised of protease and phosphatase inhibitors. The concentration of the extracted proteins was measured using the BCA Protein Quantitative Analysis Kit (Thermo, MA, USA, China). A total of 20 µg lysates were loaded onto sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels and transferred to nitrocellulose (NC) membranes (Sigma, USA). The membranes were blocked with 5% skim milk for 2 hours, and subsequently incubated with primary antibodies overnight. After incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies for 3 hours, protein bands were detected with Immobilon Western Chemiluminescent HRP Substrate (Beyotime Biotechnology, China). Densitometric quantification was carried out using ImageJ software version 1.46r (ImageJ, USA).
Immobilon western chemiluminescent hrp substrate
Manufactured by Beyotime
Sourced in China
Immobilon Western Chemiluminescent HRP Substrate is a lab equipment product that provides a chemiluminescent detection system for Western blotting applications. It is designed to detect horseradish peroxidase (HRP) conjugated to antibodies.
Automatically generated - may contain errors
Lab products found in correlation
Ripa lysis buffer, by Beyotime (2 mentions)
Bca protein quantitative analysis kit, by Thermo Fisher Scientific (1 mentions)
Nitrocellulose nc membrane, by Merck Group (1 mentions)
Ab28373, by Abcam (1 mentions)
Ab81271, by Abcam (1 mentions)
Minute plasma membrane protein isolation and cell fractionation kit, by Invent Biotechnologies (1 mentions)
Iseq00010, by Merck Group (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Beclin 1 antibody, by Cell Signaling Technology (1 mentions)
β actin antibody, by ABclonal (1 mentions)
Zinc oxide nanoparticles zno nps, by Merck Group (1 mentions)
Cy3 affinipure goat anti rabbit igg h l, by EarthOx (1 mentions)
Chemidoc xr, by Bio-Rad (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
6 protocols using immobilon western chemiluminescent hrp substrate
1
Western Blot Analysis of Proteins
2
Quantification of Hippocampal NR2B Receptors
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After hippocampal extraction, plasma membrane protein was isolated using a Minute Plasma Membrane Protein Isolation and Cell Fractionation Kit (SM-005-4; Invent Biotechnologies, Inc., Plymouth, MN, USA). Equal amounts of protein samples were separated via sodium dodecyl sulfate–polyacrylamide gel electrophoresis and subsequently transferred to polyvinylidene fluoride membranes (ISEQ00010; Millipore, Billerica, MA, USA). After being blocked for 1 hour, the membranes were incubated overnight at 4°C with the appropriate primary antibodies—NR2B (ab28373, 1:500; Abcam, Cambridge, UK), phosphorylated (p)-NR2B (ab81271, 1:300; Abcam), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; CW0100 M, 1:2000; CoWin Biosciences, Taizhou, China)—followed by a 2-hour incubation at room temperature with secondary antibodies (goat anti-rabbit IgG, CW0103S, and goat anti-mouse IgG, CW0102S; both CoWin Biosciences). Immobilon Western Chemiluminescent HRP Substrate (P0018 M; Beyotime Biotechnology, Shanghai, China) was used to detect the bound antibodies. ImageJ software (NIH, Bethesda, MD, USA) was used to quantify band intensities. The NR2B and p-NR2B band intensities were normalized to the band intensity of GAPDH, which was used as a loading control.
3
Western Blot Analysis of Mes13 Cells
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Total protein was extracted from Mes13 cells by RIPA lysis buffer (Beyotime Institute of Biotechnology) and quantitively determined using BCA Protein Assay kit (Beyotime Institute of Biotechnology). Proteins (50 µg/lane) were loaded and separated by SDS-PAGE on 10% gels and were then transferred onto polyvinylidene difluoride membranes. Membranes were blocked with non-fat milk (5% in 0.1% Tween20-PBS buffer) for 1 h at room temperature and incubated with primary antibodies (1:1,000) overnight at 4˚C. Subsequently, membranes were incubated with HRP-conjugated secondary antibodies (1:500) for 1 h at room temperature. The bands were detected using Immobilon Western Chemiluminescent HRP Substrate (Beyotime Institute of Biotechnology) and band intensities were semi-quantified using ImageJ 1.48 (National Institutes of Health).
4
Protein Expression Analysis of E. piscicida
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E. piscicida WT and mutants were statically cultured at 30°C for 14 h. The supernatants from identical amounts of bacteria were separated by SDS−PAGE and subsequently silver stained (Yeasen, China). The gel was imaged by a scanner (Epson V500, Japan).
For Western blot analysis, proteins separated by SDS-PAGE from equal amounts of bacteria were transferred onto a PVDF membrane. The membrane was then blocked with 10% nonfat milk in TBST for 2 h at room temperature. Subsequently, the membrane was probed with specific antibodies against His (Yeasen, China), RNA polymerase α subunit (Huabio, China), EseB (Huabio, China), EvpP (Huabio, China) and acetyllysine (Jingjie PTM Biolab, PTM101, China) at 4°C for 14 h. After washing with TBST three times, the membrane was incubated with the corresponding secondary antibodies at room temperature for 1 h. Following three washes with TBST, the signals on the membrane were detected with Immobilon Western Chemiluminescent HRP Substrate (Beyotime, China). The numbers correspond to the normalized protein abundance relative to the leftmost lane or DMEM group. The normalized protein abundance is the ratio of each protein band relative to the loading control, which was quantified with ImageJ.
For Western blot analysis, proteins separated by SDS-PAGE from equal amounts of bacteria were transferred onto a PVDF membrane. The membrane was then blocked with 10% nonfat milk in TBST for 2 h at room temperature. Subsequently, the membrane was probed with specific antibodies against His (Yeasen, China), RNA polymerase α subunit (Huabio, China), EseB (Huabio, China), EvpP (Huabio, China) and acetyllysine (Jingjie PTM Biolab, PTM101, China) at 4°C for 14 h. After washing with TBST three times, the membrane was incubated with the corresponding secondary antibodies at room temperature for 1 h. Following three washes with TBST, the signals on the membrane were detected with Immobilon Western Chemiluminescent HRP Substrate (Beyotime, China). The numbers correspond to the normalized protein abundance relative to the leftmost lane or DMEM group. The normalized protein abundance is the ratio of each protein band relative to the loading control, which was quantified with ImageJ.
5
Zinc Oxide Nanoparticles Biological Analysis
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Zinc oxide nanoparticles (ZnONPs), less than 50 nm particle size, were purchased from Sigma Aldrich Chemical Co. (MO, USA). Cy3 AffiniPure Goat anti-Rabbit IgG (H + L) was from EarthOx Life Sciences (CA, USA). Immobilon Western Chemiluminescent HRP Substrate, RIPA lysis buffer, phenylmethanesulfonylfluoride (PMSF) and bicinchoninic acid (BCA) assay kit were all purchased from Beyotime Institute of Biotechnology (Shanghai, China). β-actin antibody was obtained from ABclonal Biotechnology (MA, USA). Antibodies against HMG-CoA, LC3B, and p62 were all from Abcam Co. (Cambridge, UK). Beclin 1 antibody was purchased from Cell Signaling Technology (Beverly, MA, USA). GAPDH antibody was obtained from Bioss Biotechnology Co., Ltd. (Beijing, China). SREBP2 antibody was from Novus Biologicals Inc. (Littleton, CO, USA).
6
Western Blot Analysis of Fly Proteins
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Fly tissues or cell extracts were lysed in 100 mL SDS lysis buffer (20 mM Tris/HCl at pH 7.6, 150 mM NaCl, 5 mM EDTA, 10% glycerol, 1% SDS, and 1 mM PMSF) for 30 min on ice. Debris was pelleted at 16000 x g and 4 C for 10 min, then eluted in 5x SDS loading buffer and boiled for 5 min at 95 C. Samples were separated on 8%-10% SDS-PAGE and transferred onto a PVDF membrane (BIO-RAD). Membranes were blocked in 5% BSA in TBS-T, incubated overnight with the corresponding primary antibody at 4 C, followed by incubation with HRP-conjugated secondary antibodies for 2 hr at RT. Proteins were visualized using the Immobilon Western chemiluminescent HRP substrate (Beyotime) on ChemiDoc TM XRS+ (BIO-RAD). Three independent biological experiments were performed.
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