BV2 cells were harvested, washed twice with PBS, and lysed in RIPA buffer (Nanjing Jiancheng Bioengineering Institute). Protein concentrations in the lysates were quantified by the bicinchoninic acid method following the manufacturer's instructions (Nanjing KeyGen Biotech Co., Ltd.). A total of 30 µg of protein per sample was loaded and separated on 12% Mini-Protean® TGX™ gels (Bio-Rad Laboratories, Inc.) and subsequently transferred onto a polyvinylidene difluoride membrane. Prior to antibody incubations, the samples were blocked with 5% skimmed milk at room temperature for 1 h. PPM1A (dilution 1:1,000; cat. no. ab14824), JNK (dilution 1:1,000; cat. no. ab179461), phosphorylated (p)-JNK (dilution 1:3,000; cat. no. ab4821), caspase-3 (dilution 1:1,000; cat. no. ab13847), cleaved caspase-3 (dilution 1:500; cat. no. ab2302) and GAPDH (dilution 1:1,000; cat. no. ab181602) protein levels were assessed using specific antibodies (Abcam) at 4˚C overnight. Horseradish peroxidase-conjugated goat anti-mouse polyclonal antibody (dilution 1:5,000; Santa Cruz Biotechnology, Inc.; cat. no. sc-2031) was used as the secondary antibody for 30 min at room temperature. The blot was developed using Western Lightning Ultra chemiluminescent substrate (Bio-Rad Laboratories, Inc.) in an EpiChemi3 darkroom (UVP, LLC). Image Lab 3.0 software used to analyze the results (Bio-Rad Laboratories, Inc.).
Ripa buffer
Manufactured by Nanjing Jiancheng
Sourced in China
RIPA (Radio-Immunoprecipitation Assay) buffer is a commonly used detergent-based buffer solution designed for cell lysis and protein extraction. Its core function is to solubilize proteins from cells and tissues, allowing for their subsequent analysis and manipulation.
Automatically generated - may contain errors
Lab products found in correlation
Phenyl methyl sulfonyl fluoride (pmsf), by Merck Group (2 mentions)
Ecl kit, by Nanjing Jiancheng (2 mentions)
Specific antibodies, by Abcam (1 mentions)
Mini protean tgx gel, by Bio-Rad (1 mentions)
Western lightning ultra chemiluminescent substrate, by Bio-Rad (1 mentions)
Image lab 3, by Bio-Rad (1 mentions)
Anti nrf2, by Cell Signaling Technology (1 mentions)
Polyvinylidene difluoride membrane, by Cytiva (1 mentions)
Anti bcl2, by Sangon (1 mentions)
Ecl kit, by Cytiva (1 mentions)
Anti stat3, by Cell Signaling Technology (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Cell Signaling Technology (1 mentions)
T sod assay kit, by Nanjing Jiancheng (1 mentions)
Mda assay kit, by Nanjing Jiancheng (1 mentions)
Gsh px, by Nanjing Jiancheng (1 mentions)
Ros assay kit, by Nanjing Jiancheng (1 mentions)
Gsh px assay kit, by Nanjing Jiancheng (1 mentions)
Cat assay kit, by Nanjing Jiancheng (1 mentions)
Horseradish peroxidase hrp conjugated secondary antibody, by Cell Signaling Technology (1 mentions)
5 protocols using ripa buffer
1
Western Blot Analysis of BV2 Cells
2
Western Blot Analysis of Cellular Proteins
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Tissues or cells were lysed for total protein extraction in radioimmunoprecipitation assay (RIPA) buffer (Nanjing Jiancheng Bioengineering Institute, China) together with a protease inhibitor PMSF (Sigma-Aldrich Corporation, Saint Louis, MO, USA). Proteins were separated by SDS-poly-acrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Amersham Life Science, Piscataway, NJ). Membranes were incubated for 2 h in PBS blocking buffer, and then probed with antibodies. Anti-STAT3, anti-pSTAT3 (Tyr705), anti-SOCS3 and anti-Nrf2 antibodies were purchased from Cell Signaling Technology Inc., Danvers, MA; anti-G6Pase antibody was purchase from Santa Cruz; anti-PCK, anti-Bcl2 and anti-Bax antibodies were purchased from Sangon Biotech Co., Ltd, China. The membranes were washed three times for 10 min in PBST then incubated in secondary antibody diluted in TBS blocking buffer for 1 h at room temperature. Membranes were developed using the ECL reagents, Amersham (Pittsburgh, PA). Blots were developed using an ECL kit (Amersham Biosciences, Piscataway, NJ) and exposed with Blue Basic Autorad Film (ISC BioExpress).
3
Western Blot Protein Quantification Protocol
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RIPA buffer (Jiancheng, Nanjing, China) was used to extract the proteins of tissue samples and cells. The protein extracts were separated on a sodium dodecyl sulfatepolyacrylamide gels (SDS-PAGE), which were further transferred to PVDF membranes. Next, the blots were incubated in 5% nonfat dry milk in phosphate-buffered saline (PBS) with 0.1% Tween 20 for a duration of 2 hrs followed by incubating with primary antibodies (Proteintech, Wuhan, China) overnight at a temperature of 4°C. Next, the blots were incubated in horseradish peroxidase conjugated secondary antibody (Cell Signaling Technology, USA) for a duration of 2 hrs. Following this, the proteins were visualized using an ECL kit (Jiancheng, Nanjing, China), and quantified using the Image J software.
4
Antioxidant Enzyme Activity Measurement
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Liver and pancreatic tissues were lysed for total protein extraction in radioimmunoprecipitation assay (RIPA) buffer (Nanjing Jiancheng Bioengineering Institute, China) together with a protease inhibitor PMSF (Sigma-Aldrich Corporation, Saint Louis, MO, USA). ROS and MDA contents were measured by ROS Assay Kit and MDA assay kit which were purchased from Nanjing Jiancheng Bioengineering Institute, China. The activities of antioxidant enzymes, namely total superoxide dismutase (T-SOD), catalase (CAT), glutathione reductase (GR) and Glutathione peroxidase (GSH-Px), were measured by T-SOD assay kit, CAT assay kit, GR assay kit and GSH-Px assay kit which were purchased from Nanjing Jiancheng Bioengineering Institute, China.
5
Western Blot Protein Quantification
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The total protein of the samples was extracted using radioimmunoprecipitation assay (RIPA) buffer (Jiancheng, Nanjing, China). The protein extracts were separated on a sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes. The blots were incubated in 5% non-fat dry milk in phosphate-buffered saline (PBS) with 0.1% Tween 20 for 2 h followed by incubation with primary antibodies (Abcam, MA, USA) overnight at 4°C. The blots were then washed in TBST and incubated in horseradish peroxidase (HRP)-conjugated secondary antibody (Cell Signaling Technology, USA) for 2 h at room temperature. The proteins were visualized using an enhanced chemiluminescence (ECL) kit (Jiancheng) and quantified by ImageJ software.
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