Cells were lysed in lysis buffer (containing 20 mM HEPES, pH 7.4, 150 mM NaCl, 0.5% Triton X-100, and EDTA-free proteinase inhibitor cocktail (MedChemexpress, USA) and phosphatase inhibitor (Yeasen, China)) on ice. The lysates were spun down at 13,000g for 30 min at 4 °C. The resulted cell lysates were quantified using BCA kit (Thermo Scientific, USA). Proteins were resolved by SDS-PAGE and transferred to nitrocellulose membrane, and then blocked with 5% bovine serum albumin (BSA). The corresponding membranes were incubated with primary antibodies overnight at 4 °C, following the incubation with HRP-conjugated secondary antibodies. The bands were visualized by an ECL kit (Thermo Scientific, USA). The blots were quantified by analysis of the grayscale using imageJ software.
Phosphatase inhibitor
Manufactured by Yeasen
Sourced in China
Phosphatase inhibitors are chemical compounds used in laboratory settings to block the activity of phosphatase enzymes. Phosphatases are responsible for removing phosphate groups from various biomolecules, and inhibiting their function can help preserve the phosphorylation state of proteins and other molecules during experimentation and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Bca kit, by Thermo Fisher Scientific (3 mentions)
Ecl kit, by Thermo Fisher Scientific (2 mentions)
Ripa lysis buffer, by Yeasen (2 mentions)
Protease, by Yeasen (2 mentions)
Skimmed milk, by Yeasen (1 mentions)
Imagequant 400, by GE Healthcare (1 mentions)
Amersham ecl prime, by Cytiva (1 mentions)
Phenylmethylsulfonyl fluoride, by Merck Group (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Image lab 3, by Bio-Rad (1 mentions)
Protease inhibitor cocktail, by Yeasen (1 mentions)
Rabbit monoclonal anti β actin, by Cell Signaling Technology (1 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin solution, by Thermo Fisher Scientific (1 mentions)
Rabbit monoclonal anti opa1, by Cell Signaling Technology (1 mentions)
Mito tracker, by Yeasen (1 mentions)
Rabbit monoclonal anti mfn2, by Cell Signaling Technology (1 mentions)
Sds page gel preparation kit, by Yeasen (1 mentions)
Dimethyl sulfoxide (dmso), by Yeasen (1 mentions)
Trypsin edta, by Thermo Fisher Scientific (1 mentions)
6 protocols using phosphatase inhibitor
1
Western Blot Quantification Protocol
2
SDS-PAGE and Western Blot Analysis
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Cells were lysed using a radioimmunoprecipitation assay lysis buffer with 10% phosphatase inhibitor (Shanghai Yeasen Biotechnology Co., Ltd.) and 1% phenylmethylsulfonyl fluoride (Sigma-Aldrich; Merck KGaA) for 30 min at 4°C. Total protein lysate was then collected, and the concentration was determined with a BCA Protein Assay kit (Beyotime Institute of Biotechnology). After denaturation at 100°C for 5 min, 20 µg of protein samples from each group were resolved by SDS-PAGE using 10% gels, then electrophoretically transferred to a PVDF. The PVDF membrane was blocked with 5% skimmed milk (Shanghai Yeasen Biotechnology Co., Ltd.) for 1.5 h at room temperature. The membrane was incubated overnight with primary antibodies at 4°C and for 1 h with HRP-conjugated secondary antibodies (1:10,000) at 37°C (Table II ). Amersham ECL Prime (Cytiva) was used to detect bands. The densities of the specific protein bands were visualized and captured using ImageQuant™ 400 (GE Healthcare Life Sciences) and analysed using Image Lab 3.0 software (Bio-Rad Laboratories).
3
Mitochondrial Dynamics Regulation in Cell Apoptosis
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Dulbecco’s Modified Eagle’s Medium (DMEM), penicillin-streptomycin solution, 0.25% trypsin/EDTA, and phosphate-buffered saline were purchased from Gibco. Fetal bovine serum (FBS) was purchased from Biological Industries. Rabbit monoclonal anti-Mfn1(Cat#: 14739S), rabbit monoclonal anti-Mfn2(Cat#: 9482S), rabbit monoclonal anti-Opa1(Cat#: 80471S), rabbit polyclonal anti-Drp1(Phospho Ser616 Cat#: 4494S), rabbit monoclonal anti-AMPKα (Phospho Thr172 Cat#: 2535S), and rabbit monoclonal anti-β-actin(Cat#: 4970T) were purchased from Cell Signaling Technology. Rabbit monoclonal anti-Fis1(Cat#: ab156865) and rabbit monoclonal anti-DRP1(Cat#: ab184247) were purchased from Abcam. DMSO, Mito-Tracker, BCA Protein Quantification Kit, RIPA lysis buffer, protease inhibitor cocktail, phosphatase inhibitors, SDS-PAGE Gel Preparation Kit, and other Western blotting reagents were purchased from Yeasen (Shanghai, China). Annexin V-Alexa Fluor 647/PI Apoptosis Detection Kit was purchased from Fcmacs (Nanjing, China). The mitochondrial membrane potential assay kit with JC-1 was purchased from Beyotime (Nanjing, China). Unless otherwise indicated, all other chemicals and reagents were purchased from Sigma-Aldrich.
4
Optimized Protein Extraction and Detection Workflow
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The cells were washed using an ice-cold phosphate-buffered saline (PBS) and then lysed with the Membrane and Cytosol Protein Extraction Kit (20127ES60 Yeasen, China). The kit was further supplemented with protease (20124ES03 Yeasen, China) and phosphatase inhibitors (20109ES05 Yeasen, China). The concentration of the proteins was determined via the BCA Protein Assay Kit (20201ES76 Yeasen, China), and the process was conducted in accordance with the manufacturer’s instructions. The proteins were segregated on 4–20% Bis-Tris gels (Genscript, China), subsequently being transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore ISEQ00010, China). The membranes were then blocked using 5% non-fat milk in Tris-buffered saline with Tween 20 (TBST) at room temperature for 1 hour. Primary antibodies were diluted in 0.5% non-fat milk in TBST and were then allowed to incubate with the membrane at 4°C overnight: Anti-LTBR antibody (Absin abs146148, 1:1,000), Anti-GAPDH (Absin abs830030, 1:2,000). After three washes with TBST, the membrane was incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (34201ES60 Yeasen, China) in TBST at room temperature for 1 hour. The immunoreactive bands were then visualized utilizing the Enhanced Chemiluminescence (ECL) Western Blotting Substrate (36208ES60 Yeasen, China).
5
Protein Expression Analysis by Western Blot
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The cells were lysed in Radio Immunoprecipitation Assay (RIPA) Lysis buffer supplemented with proteases (Yeason, China) and phosphatase inhibitors (Yeason, China) for 20-min incubation on ice. The bicinchoninic acid assay kit (Yeason, China) was conducted to measure the protein concentration in cell lysates. About 8%–12% SDS-polyacrylamide gels were used to separate total protein lysates from different samples, then Polyvinylidene fluoride (PVDF) membranes were electrophoretically transferred (130 V, 50 min at 4°C). The blocking solution was applied to PVDF membranes followed by incubation with specific antibodies overnight at 4°C. The following antibodies were used in this study: anti-β-actin (1:4000, ab8227, Abcam), anti-MMP9 (1:1000, CST, Massachusetts, United States), anti-MMP2 (1:1000, CST, Massachusetts, United States), anti-MEK (1:1000, CST, Massachusetts, United States), anti-p-MEK (1:1000, CST, Massachusetts, United States), anti-p-ERK (1:1000, CST, Massachusetts, United States), anti-ERK (1:1000, CST, Massachusetts, United States), anti-GPX4 (1:1000, CST, Massachusetts, United States).
6
Protein Extraction from Cells and Tissues
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All protein extraction procedures were performed on ice. The cells protein was extracted using a mixture of T-PER Protein Extraction Reagent lysates (Thermo Fisher Scientific, USA) plus protease (Yeasen, China) and phosphatase inhibitors (Yeasen, China) according to the manufacturer's instructions, and then incubated on ice for 30 min. For tissue samples protein extraction, tissue samples were thawed on ice and ground in liquid nitrogen, then dissolved on ice for 45 min. Both lysates were centrifuged with a speed of 12000 rpm for 15 min at 4°C, and the supernatant was collected and frozen at -80 °C. A bicinchoninic acid (BCA) protein assay kit (Pierce, USA) was used to determine the protein concentrations according to the manufacturer's instructions. PBS was used as the standard.
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