Western blotting was performed as previously described 33 (link). In brief, cell lysates were prepared by RIPA buffer (Beyotime, shanghai, China) supplemented with a PMSF inhibitor (Beyotime, shanghai, China), and quantitative analyzed with a BCA kit (Thermo scientific, MA, USA). Protein extracts were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a polyvinylidene difluoride (PVDF) membrane filter. After incubation with the desired antibodies, the blots were developed with Thermo Scientific's SuperSignal West Pico Chemiluminescent substrate or Millipore's Immunobilon Western Chemiluminescent HRP substrate.
Immunobilon western chemiluminescent hrp substrate
Manufactured by Merck Group
Sourced in United States
Immunobilon Western Chemiluminescent HRP Substrate is a laboratory reagent used to detect and quantify proteins in Western blot analysis. It is a chemiluminescent substrate for horseradish peroxidase (HRP) enzyme, which is commonly used to label target proteins in Western blotting experiments.
Automatically generated - may contain errors
Lab products found in correlation
Bca kit, by Thermo Fisher Scientific (3 mentions)
Polyvinylidene difluoride membrane, by Merck Group (2 mentions)
Secondary antibody conjugated to horseradish peroxidase, by Thermo Fisher Scientific (2 mentions)
Supersignal west pico chemiluminescent substrate, by Merck Group (1 mentions)
Pmsf inhibitor, by Beyotime (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
Protease inhibitor mixture, by Roche (1 mentions)
Lysis buffer, by Cell Signaling Technology (1 mentions)
Imagequant digital imager, by GE Healthcare (1 mentions)
Bca assay, by Thermo Fisher Scientific (1 mentions)
β actin, by Merck Group (1 mentions)
Image station 4000mm, by Kodak (1 mentions)
Ab2786, by Abcam (1 mentions)
Ab184154, by Abcam (1 mentions)
Ab119871, by Abcam (1 mentions)
Ab224722, by Abcam (1 mentions)
Ab25377, by Abcam (1 mentions)
Ab134047, by Abcam (1 mentions)
Beclin 1, by Cell Signaling Technology (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
6 protocols using immunobilon western chemiluminescent hrp substrate
1
Western Blot Protocol for Protein Analysis
2
Western Blot Protein Expression Analysis
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Cells were incubated in lysis buffer (Cell Signaling Technology) containing protease inhibitor mixtures (Roche Molecular Biochemicals, Indianapolis, IN) and phenylmethylsulfonyl fluoride (PMSF) at 4 °C for 30 min. The protein concentration was determined using bicinchoninic acid (BCA) assay (Pierce Biotechnology, Rockford, IL, USA). Proteins (30 − 60 µg) were separated by 12% SDS-PAGE and transferred onto PVDF membranes. Membranes were blocked with 5% non-fat milk in TBST for 1 h at room temperature and incubated with appropriate primary antibodies overnight at 4 °C, followed by secondary antibodies for 1 h at room temperature. Immunoblots were detected by Immunobilon Western Chemiluminescent HRP substrate (Millipore, Burlington, MA) and visualized on an ImageQuantTM digital imager (GE Healthcare, Pittsburgh, PA).
3
Western Blot Analysis of TRPC5 in Neurons and Astrocytes
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Membrane proteins from cultured pure neurons and astrocytes were extracted with lysis buffer (20 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.5% SDS, 2.5 mM sodium pyrophosphate, 1 μM NaVO4, and protease inhibitors). Proteins were separated by 8% SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Millipore). Membranes were probed with antibodies to TRPC5 (Neuromab, 75-104) and β-actin (Sigma, A0560), followed by incubation with the appropriate secondary antibody conjugated to horseradish peroxidase (Thermo Fisher Scientific). Immunoreactivity was visualized using Immunobilon™ Western Chemiluminescent HRP Substrate (Millipore) and the Kodak Image Station 4000MM (Kodak).
4
Western Blot Analysis of Cardiac Proteins
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Cardiac tissue samples for western blotting were obtained from the infarcted area. Proteins isolated from sI/R-treated CMECs were also analyzed through western blots. In brief, proteins were separated by SDS electrophoresis and transferred to membranes using standard protocols,67 (link) after which they were probed with antibodies against p-eNOS (Ser1117) (1:1,000, #ab184154; Abcam), ET-1 (1:1,000, #ab2786; Abcam), ICAM1 (1:1,000, #ab119871; Abcam), VCAM1 (1:1,000, #ab134047; Abcam), Gr1 (1:1,000, #ab25377; Abcam), FUNDC1 (1:1,000, #ab224722; Abcam), ATG5 (1:1,000, #12994, Cell Signaling Technology), and Beclin1 (1:1,000, #3738; Cell Signaling Technology). The blots were visualized by chemiluminescence (ECL, Immunobilon Western Chemiluminescent HRP Substrate, Millipore Corporation, MA, USA), and the signals were quantified by densitometry. GAPDH (analyzed with an antibody from Santa Cruz Biotechnology) served as a loading control.68 (link)
5
PTEN and GAPDH Protein Analysis
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CCRCC cells were lysed in buffer containing 50 mM Tris–HCl (pH 7.5), 150 mM NaCl, 0.1% (w/v) SDS, 0.1% (w/v) SDC, 1% (v/v) Nonidet P-40, 5 mM NaF, 1 mM Na3VO4, 2.5 mM Na2HPO4, and protease inhibitor cocktail (Roche Diagnostics, Basel, Switzerland) and tissues were lysed in Pro-prep buffer (Intron, Daejeon, Korea). Equal amounts of proteins were separated by SDS–polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA). Membranes were probed with antibodies to PTEN (Cell Signaling, 91885) and GAPDH (sc-20357, Santa Cruz Biotechnology, Inc.), followed by incubation with the appropriate secondary antibody conjugated to horseradish peroxidase (ThermoFisher Scientific, Waltham, MA). Immunoreactivity was visualized using Immunobilon Western Chemiluminescent HRP Substrate (Millipore) and the Amersham Imager 600.
6
Western Blot Analysis of Protein Signaling
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Cell proteins were extracted using M-PER Mammalian Protein Extraction Reagent (Thermo Fisher Scientific, Waltham, MA, USA), supplemented with protease inhibitor and phosphatase inhibitor (Sigma, St. Louis, MO, USA). Western blots were prepared by standard procedures using anti-DDK (Origene Tech, Rockville, MD, USA), anti-actin (Santa Cruz, CA, USA), anti-AKT, anti-phospho-AKT, anti-mTOR, and anti-phospho-mTOR (Cell signaling Technology, Danvers, MA, USA). Immunoreactivity was detected by an Immunobilon Western Chemiluminescent HRP Substrate (Millipore, Billerica, MA, USA).
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