Cell lysates were prepared in RIPA buffer containing Halt Protease and Phosphatase Inhibitors Cocktail (Pierce Biotechnology Inc.), and centrifuged at 4,390 g for 10 minutes at 4°C. Protein concentration was measured using a Bio-Rad Protein Assay (Bio-Rad Laboratories). Equal amounts of proteins (10–15 μg) from each sample were subjected to SDS/PAGE and transferred onto nitrocellulose membranes (Whatman, Boston, MA). Membranes were first probed with primary antibodies and subsequently with HRP-conjugated secondary antibodies. Protein bands detected by a commercial Immobilon Western Chemiluminescent HRP Substrate detection reagent (Millipore).
Immobilon western chemiluminescent hrp substrate detection reagent
Manufactured by Merck Group
Sourced in United States
Immobilon Western Chemiluminescent HRP Substrate detection reagent is a laboratory product designed for the detection of horseradish peroxidase (HRP) in western blot analyses. It is a chemiluminescent substrate that emits light upon reaction with HRP, enabling the visualization and quantification of target proteins.
Automatically generated - may contain errors
Lab products found in correlation
Protein assay, by Bio-Rad (2 mentions)
Image lab software, by Bio-Rad (2 mentions)
Immobilon pvdf membrane, by Merck Group (2 mentions)
Dynabeads protein a immunoprecipitation kit, by Thermo Fisher Scientific (2 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
Nitrocellulose membrane, by Cytiva (1 mentions)
Halt protease and phosphatase inhibitors cocktail, by Thermo Fisher Scientific (1 mentions)
Chemidoc xrs system, by Bio-Rad (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Protran nitrocellulose membrane, by Cytiva (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Cell Signaling Technology (1 mentions)
Mitochondria isolation kit, by Solarbio (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Chemidoc imaging system, by Bio-Rad (1 mentions)
Bicinchoninic acid protein assay kit, by Beyotime (1 mentions)
11 protocols using immobilon western chemiluminescent hrp substrate detection reagent
1
Cell Lysis and Western Blot
2
Protein Expression Analysis via Western Blot
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The protein extraction procedures were similar to Section 2.10. The equivalent protein samples were subjected to 12% SDS-PAGE followed by western blot analysis. The PVDF membranes were blocked by 5% skim milk in TBST for 1 h at room temperature and then were incubated overnight at 4°C by the primary antibodies: anti-human PKM (1 : 1000), ATP8 (1 : 1000), ITGB3 (1 : 2000), GATM (1 : 1000), SERPINE1 (1 : 2000), PCK2 (1 : 1000), and β-Actin (1 : 1000). After primary antibodies incubation, the PVDF membranes were incubated with HRP-conjugated secondary antibodies for 1 h at room temperature. The proteins were stained with Immobilon™ Western Chemiluminescent HRP Substrate detection reagent (Millipore, MA, USA) and captured using Image Lab™ software (Bio-Rad, VA, USA). To quantify the relative intensity of proteins, the ratios of target proteins to β-actin signals were calculated by ImageJ software.
3
Immunoprecipitation and Western Blotting
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Cells were lysed with TENN buffer containing 0.5% Triton X-100, 150 mmol/L NaCl, 5 mmol/L EDTA, and 50 mmol/L Tris supplemented with protease and phosphatase inhibitors (Rockford, IL). Immunoprecipitations of equal total protein amounts were performed using Dynabeads® Protein A Immunoprecipitation Kit (Invitrogen) following the manufacturer’s protocol. Briefly, protein samples were incubated with primary antibody-bound Protein A Dynabeads. The beads were washed four times and heated to 100°C for 5 min in 20 μl of sample buffer. Proteins were separated on 12% SDS-PAGE and transferred to Immobilon PVDF membrane (Millipore). Membranes were probed with specific antibodies. Proteins were then visualized by using horseradish peroxidase (HRP)-conjugated secondary antibodies and Immobilon Western Chemiluminescent HRP Substrate detection reagent (Millipore). β-actin was used as loading control.
4
Immunoprecipitation and Western Blotting
5
Western Blotting Protocol for Protein Detection
6
Western Blot Protein Extraction
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Total protein was extracted using a lysis buffer containing 50 mM Tris–HCl (pH 7.4), 150 mM NaCl, 1 % Triton X-100, 0.1 % SDS, and 1 mM EDTA supplemented with protease inhibitors and phosphatase inhibitors provided by Sigma. The protein extract was loaded, size-fractionated by SDS–polyacrylamide gel electrophoresis, and transferred to PVDF membranes (Bio-Rad). After blocking, the membranes were incubated with primary antibodies at 4 °C overnight. Membranes were probed with specific antibodies, and proteins were visualized by using HRP-conjugated secondary antibodies and Immobilon Western Chemiluminescent HRP Substrate Detection Reagent (Millipore, Billerica, MA, USA). Gel loading was normalized for equal β-actin.
7
Western Blot Protein Analysis Protocol
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Cells were lysed in RIPA buffer (50 mM Tris-HCl buffer, pH 7.4, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, and 0.1% SDS) supplemented with 1 × Halt protease inhibitor cocktail and 1 × Halt phosphatase inhibitor cocktail (Pierce, Rockford, IL). The Bio-Rad Protein Assay (Bio-Rad, Hercules, CA) was utilized to determine protein concentrations prior to analysis. Proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to Protran nitrocellulose membranes (Whatman, Boston, MA). Membranes were probed with specific primary antibodies (1:1000 dilution) and horseradish peroxidase-conjugated secondary antibodies (Cell Signaling). Protein bands were visualized using Immobilon Western Chemiluminescent HRP Substrate Detection Reagent (Millipore, Billerica, MA). Gel loading was normalized for equal β-actin.
8
Lung Tissue Protein Extraction and Western Blotting
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The protein samples were extracted by lysing 1 mg of lung tissues in 1 mL of ice-cold RIPA buffer for 30 min and centrifugated at 20,000 rpm, 4°C for 15 min. The protein extractions were boiled at 100°C for 5 min before western blotting. 20 µg protein of each sample was subjected to 12% SDS-PAGE and then transferred to PVDF membranes. The membranes were blocked by 10% goat serum and then incubated with primary antibodies diluted in TBST: AP-1 (1 : 2000), β-actin (1 : 2000), p-NF-κBp65 (Ser536) (1 : 1000), NF-κBp65 (1 : 1000), p-IκBα (Ser32) (1 : 1000), and IκBα (1 : 1000), overnight at 4°C. The next day, PVDF membranes were incubated with HRP-conjugated secondary antibodies at room temperature for 2 h, and then, proteins were visualised by Immobilon Western Chemiluminescent HRP Substrate detection reagent (Millipore, Bedford, MA, USA).
9
Mitochondrial Protein Extraction and Western Blot
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Total protein was extracted from cancer cells using Mammalian Protein Extraction Buffer (P0013, Beyotime, Beijing, China) supplemented with protease inhibitor cocktail (87786, ThermoFisher, USA). The mitochondrial fractions were separated using Mitochondria Isolation Kit (number: SM0020, Solarbio, Beijing, China) according to the manufacturer’s protocol, and the protein was extracted as above. Equal amounts of protein lysates were separated by SDS-PAGE gel and electro-transferred to PVDF membrane (Millipore, USA). After blocking in 5% milk-PBST for 2 h at 37 °C, the membranes were incubated overnight with primary antibodies (Additional file 2 ) at 4 °C and with secondary antibodies at 37 °C for 2 h. The positive bands were visualized with Immobilon Western Chemiluminescent HRP Substrate detection reagent (Millipore, USA), and acquired using a ChemiDoc™ imaging System (Bio-Rad, USA).
10
Protein Extraction and Western Blot Analysis
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Total proteins in cells were extracted by ice cold RIPA lysis buffer added PMSF for 30 min. While hepatic protein extractions were performed by rstly pulverizing hepatic tissues in liquid nitrogen and lysing the fragments in ice cold RIPA buffer for 30 min. The protein extractions were centrifugated at 15000 rpm at 4 °C for 10 min and the supernatant were quanti ed by bicinchoninic acid protein assay kit (Thermo Fisher Scienti c). The equivalent protein samples were subjected to 10% SDS-PAGE followed by western blot analysis. The PVDF membranes were blocked by 5% skim milk in TBST for 1 h at room temperature, and next were incubated overnight at 4 °C by the primary antibodies diluted in TBST: anti-NFAT2
(1:1000), Egr2 (1:1000), FasL (1:2000), AKT (1:1000), p-AKT (1:1000), ERK (1:1000), p-ERK (1:1000), COX-2 (1:1000), c-myc (1:2000), and β-Actin (1:1000). After primary antibodies incubation, the PVDF membranes were incubated with HRP-conjugated secondary antibodies for 1 h at room temperature. The proteins were stained with Immobilon™ Western Chemiluminescent HRP Substrate detection reagent (Millipore, MA, USA) and captured using Image Lab™ software (Bio-Rad, VA, USA). To quantify the relative intensity of proteins, the ratios of target proteins to β-actin signals were calculated by ImageJ software.
(1:1000), Egr2 (1:1000), FasL (1:2000), AKT (1:1000), p-AKT (1:1000), ERK (1:1000), p-ERK (1:1000), COX-2 (1:1000), c-myc (1:2000), and β-Actin (1:1000). After primary antibodies incubation, the PVDF membranes were incubated with HRP-conjugated secondary antibodies for 1 h at room temperature. The proteins were stained with Immobilon™ Western Chemiluminescent HRP Substrate detection reagent (Millipore, MA, USA) and captured using Image Lab™ software (Bio-Rad, VA, USA). To quantify the relative intensity of proteins, the ratios of target proteins to β-actin signals were calculated by ImageJ software.
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