Cells were treated according to different needs of the experiment and lysed with RIPA (Beyotime) on ice to collect cell proteins. The protein expression of the cells was then detected by western blot technology. The antibodies needed for the proteins detected in this experiment are: anti-RBCK1 (26367-1-AP, Proteintech), anti-HIF1α (SC-135151, Santa Cruz), anti-β-Actin (A5441, Sigma), anti-HA (MMS-101R, Biolegend), anti-Myc (60003-2-lg, Proteintech), and anti-Flag (20543-1-AP, Proteintech). After the protein was electrophoretic, transparabed, and blocked, we incubated the corresponding primary antibody and the secondary antibody of the primary antibody species. Finally, we visualized the fluorescent signal of the resulting protein using AI600 (GE), during which the membrane was pre-processed with an Immobilon Western Chemilum HRP Substrate Kit (Millipore Co, Billerica).
Immobilon western chemilum hrp substrate kit
Manufactured by Merck Group
Sourced in United States
The Immobilon Western Chemilum HRP Substrate Kit is a laboratory product used for the detection and visualization of proteins in Western blot analyses. It provides a chemiluminescent substrate for the horseradish peroxidase (HRP) enzyme, which is commonly used as a detection label in immunoassays.
Automatically generated - may contain errors
Lab products found in correlation
Ai600, by GE Healthcare (2 mentions)
Image lab software, by Bio-Rad (2 mentions)
Radioimmunoprecipitation assay (ripa), by Beyotime (1 mentions)
Anti rbck1 26367 1 ap, by Proteintech (1 mentions)
Anti β actin a5441, by Merck Group (1 mentions)
Anti flag 20543 1 ap, by Proteintech (1 mentions)
St506 p0013, by Beyotime (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Lysis buffer, by Beyotime (1 mentions)
Phenylmethanesulfonyl fluoride, by Beyotime (1 mentions)
Bicinchoninic acid protein assay kit, by Beyotime (1 mentions)
Gel doc xr imaging system, by Bio-Rad (1 mentions)
Bca protein assay kit, by Solarbio (1 mentions)
Ripa buffer, by Solarbio (1 mentions)
Hrp conjugated anti mouse igg, by Merck Group (1 mentions)
Hrp conjugated anti rabbit igg, by Merck Group (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
6 protocols using immobilon western chemilum hrp substrate kit
1
Protein Expression Analysis via Western Blot
2
Detection of HIF1α K48 Polyubiquitination
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To detect the K48 polyubiquitination of HIF1α in cells, we co-transfected Flag-RBCK1/Flag-tag and Myc-HIF1α and K48-Ub plasmids in HEK293T. 48 h after transfection by a western blot and IP technology, we obtained the corresponding protein supernatant. Finally, we visualized the fluorescent signal of the resulting protein using AI600 (GE), during which the membrane was pre-processed with an Immobilon Western Chemilum HRP Substrate Kit (Millipore Co, Billerica).
3
Proteasome Inhibitor-based Protein Extraction
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Cell proteins were collected with Western and IP lysates. A proteasome inhibitor was also used (ST506 p0013, Beyotime). 12000×G was kept at 4 °C after centrifugation for 30 min, and the collected supernatant was incubated with the required antibody or control IgG and protein A/G agarose (p2051 p2053, Beyotime) at 4 °C overnight. The next day, at 4 °C, 3000 × G thrived thrice through centrifugation for 10 min and rinsed with a lysis buffer (p0013f, Beyotime). The supernatant was discarded. A 2×SDS-PAGE buffer was added and boiled at 99 °C for 10 min. SDS-PAGE electrophoresis was performed. The resulting membrane was then incubated with the corresponding primary antibody overnight at 4 °C. After membrane washing, it was incubated with the secondary antibody of HRP-labeled Goat anti-Mouse/Rabbit IgG (H + L) for 2 h. Finally, we visualized the fluorescent signal of the resulting protein using AI600 (GE), during which the membrane was pre-processed with the Immobilon Western Chemilum HRP Substrate Kit (Millipore Co, Billerica).
4
Western Blot Analysis of Drug Effects
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Western blotting analysis was performed as previously described.11 After drug treatment for 24 hours, cells were washed with PBS buffer and lysed in radioimmunoprecipitation assay Lysis Buffer (Beyotime, Shanghai, People’s Republic of China) containing 1% dilution of the phenylmethanesulfonyl fluoride (Beyotime) on ice. Protein concentration was determined by bicinchoninic acid protein assay kit (Beyotime) according to the manufacturer’s protocol. Equal amounts of protein samples (30–50 μg) were separated by 8% SDS-polyacrylamide gel electrophoresis and transferred onto a polyvinylidene fluoride membrane (Merck Millipore, Billerica, MA, USA). After blocking in 5% non-fat milk, the membranes were incubated with the specific primary antibodies at 4°C overnight. Then, the membranes were incubated with horseradish peroxidase labeled goat anti-mouse or anti-rabbit secondary antibody at room temperature for 1 hour. Finally, proteins were detected with an Immobilon Western Chemilum HRP Substrate kit (Millipore) and were semi-quantified by Image Lab software (version 3.0; Bio-Rad Laboratories, Inc., Hercules, CA, USA). β-actin was used as a control for equal loading of samples.
5
Western Blot Analysis of EMP3 in GL261 Cells
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Protein was extracted from GL261 cells using lysis buffer containing RIPA buffer (Solarbio, 89,901) and a protease inhibitor (Solarbio, A8260). The BCA Protein Assay Kit (Solarbio, PC0020) was used to test the protein concentration according to the manufacturer’s instructions, and 20 µg/lane protein was loaded per well. The primary antibodies used were anti-EMP3 (1:1000, Abcam, 236,671) and anti-β-actin (1:1000, CST, 4970). PVDF membranes were incubated with primary antibodies overnight at 4 °C and then incubated with HRP-conjugated anti-rabbit IgG (1:10,000, Sigma-Aldrich, RABHRP1) or HRP-conjugated anti-mouse IgG (1:10,000, Sigma-Aldrich, RABHRP2) at 37 °C for 1 h. The protein bands were visualized using an Immobilon Western Chemilum HRP Substrate kit (Millipore, WBKLS0050) and Bio-Rad Gel Doc XR imaging system (Bio-Rad, USA).
6
Protein Extraction and Western Blot Analysis
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The cell precipitate was lysed with RIPA (C5029, Bioss) to extract the total protein. The protein concentration was measured using the BCA method. Around 25 μg of total protein underwent gel electrophoresis and was transferred to a PVDF membrane (Millipore, Kenilworth, NJ, USA). Afterwards, the membrane was blocked with Protein-Free Rapid Blocking Buffer (Yamei, Shanghai, China) at room temperature for 15 min. The membrane was then incubated overnight at 4 °C with the primary antibody. The next day, the membrane was washed three times with TBST for 5 min each, and then incubated with the secondary antibody and membrane at room temperature for 1 h. Detection was carried out using the Immobilon Western Chemilum HRP Substrate Kit (WBKL S0500, Millipore, Kenilworth, NJ, USA). Specific areas of the gel were scanned to acquire images, and Adobe Photoshop software (CS4, Adobe Systems, USA) and ImageLab software (Bio-Rad, USA) were used for quantification.
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