NPCs were lysed with RIPA lysis buffer (Fudebio, Hangzhou, China) containing PMSF (Fudebio, Hangzhou, China) in an ice bath. After boiling with loading buffer (Fudebio), proteins from the samples were electrophoresed using SDS–PAGE gels at 80 V for 2 h and transferred to 0.22 μm polyvinylidene difluoride (PVDF) membranes (Millipore, MA, USA) at 280 mA for 120 min. The membranes were blocked with 5% skim milk powder in TBST for 2 h at room temperature and incubated with antibodies specific for β-actin (AF5001, Beyotime), MCM7 (A11325, Abclonal, Wuhan, China), p21 (27296-1-AP, Proteintech, Wuhan, China), p16 (A0262, Abclonal), aggrecan (13880-1-AP, Proteintech), collagen II (28459-1-AP, Proteintech), MMP-13 (18165-1-AP, Proteintech), and MMP-3 (17873-1-AP, Proteintech) overnight at 4 °C. The next day, the PVDF membranes were washed with TBST and incubated with an HRP-conjugated secondary antibody (Fudebio). After washing with TBST, bands were detected using a ChemiDoc Touch Imaging System (Bio–Rad, Hercules, CA, USA) with a chemiluminescence kit (Fudebio).
Ripa lysis buffer
Manufactured by Fudebio
Sourced in China
RIPA lysis buffer is a commonly used solution for extracting proteins from cells and tissues. It contains a combination of detergents and other agents that disrupt cell membranes and solubilize proteins. The buffer is designed to maintain the native structure and function of proteins during the lysis process.
Automatically generated - may contain errors
Lab products found in correlation
Bca kit, by Thermo Fisher Scientific (3 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Chemiluminescence kit, by Fudebio (1 mentions)
A0262, by ABclonal (1 mentions)
Af5001, by Beyotime (1 mentions)
Chemidoc touch imaging system, by Bio-Rad (1 mentions)
Ipvh00010, by Merck Group (1 mentions)
Atg7 rabbit polyclonal antibody, by Thermo Fisher Scientific (1 mentions)
Prestin goat polyclonal antibody, by Santa Cruz Biotechnology (1 mentions)
Gapdh mouse monoclonal antibody, by Abcam (1 mentions)
Imagequant las 4000, by GE Healthcare (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Gp62 c, by Progen Biotechnik (1 mentions)
Protein phosphatase inhibitor, by Roche (1 mentions)
Anti cd206, by Affinity Biosciences (1 mentions)
Horseradish peroxidase hrp conjugated secondary antibodies, by Affinity Biosciences (1 mentions)
Anti cd68, by Affinity Biosciences (1 mentions)
Anti inos, by Affinity Biosciences (1 mentions)
Protease and phosphatase inhibitors, by Fudebio (1 mentions)
Enhanced chemiluminescence system, by Merck Group (1 mentions)
7 protocols using ripa lysis buffer
1
Western Blot Analysis of Cellular Proteins
2
GSDME-N Terminal Protein Detection
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T cells were lysated in RIPA lysis buffer (Fudebio-tech, FD009). Equal amounts of protein were resolved by 10 % SDS-PAGE. After electrophoresis, separated proteins were transferred onto PVDF membranes (Millipore, IPVH00010). The membrane was blocked in 5 % skim milk, followed by overnight incubation with GSDME-N terminal antibody (Abcam, ab215191, 1:1000). After incubation with HRP-conjugated secondary antibody, the positive immune reactive signal was detected using FDbio-Dura ECL Kit (Fdbio-tech, FD8020). β-actin was used as an endogenous control.
3
Western Blot Analysis of Autophagic Markers
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Cochleae and other organs were dissected and transferred to the homogenizer tubes and mixed with RIPA lysis buffer (Fudebio, FD008) with a protease inhibitor cocktail (Roche, 11697498001). The supernatant from centrifuged homogenates was mixed with sodium dodecyl sulfate buffer (Beyotime, P0015L), boiled, electrophoresed, and blotted onto a 0.2-μm polyvinylidene difluoride membrane (Millipore, Immobilon ISEQ00010). The primary antibodies were: Atg7 rabbit polyclonal antibody (Thermo Fisher, PA5-35203, 1:500), Prestin goat polyclonal antibody (Santa Cruz, sc-22694, 1:400), GAPDH mouse monoclonal antibody (Abcam, ab9484, 1:1000), β-Actin rabbit IgG antibody (Abmart, P30002, 1:1000), LC3 rabbit polyclonal antibody (CST, 4108, 1:1000), and P62 Guinea Pig polyclonal antibody C-terminal specific (Progen, GP62-C, 1:500). The ECL Kit and horseradish peroxidase-conjugated antibodies were used for detection. Images were obtained by GE ImageQuant LAS4000.
4
Protein Expression Analysis in Ovarian Cells
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Total protein of the cultured cells and mouse ovaries were harvested with RIPA lysis buffer (Fude Bio, Hangzhou, China). The protein concentration was determined using a BCA kit. The protein components were separated using SDS-PAGE and transferred to polyvinylidene fluoride membranes. After blocking with 5% skim milk for 1 h, the membranes were incubated with β-actin, LHCGR, UBE2E3, FSHR, and CCND2 antibodies overnight at 4 °C and then incubated with a secondary antibody labeled with horseradish peroxidase for 1 h at room temperature. Finally, enhanced chemiluminescence (ECL) reagent was used to detect and analyze the immunoreactive bands. Antibody information is listed in Table 1 .
5
Western Blot Analysis of Testicular Proteins
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The cells and testicular tissues were lysed in RIPA lysis buffer (Fude Bio, China) with 1 mM PMSF and protein phosphatase inhibitor (Roche, USA), and protein concentration was detected by BCA. Thirty micrograms of protein were electrophoresed on 12% SDS-PAGE and then transferred onto PVDF membranes. SF-1, CYP11A1, StAR, FoxO1, AKT, p-AKT, CCND1, CCND2, SMURF2, METTL3, METTL14, and ALKBH5 antibodies were added and incubated overnight at 4°C, and the blots were incubated with a secondary antibody labeled with horseradish peroxidase (HRP) for 1 h. Finally, the immunoreactions were detected by enhanced chemiluminescence (ECL) reagent. Antibody information is listed in Table S1 .
6
Western Blot Analysis of Macrophage Markers
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We used the RIPA Lysis Buffer (Fudebio, Hangzhou, China) containing phosphatase/protease inhibitors (Fudebio, Hangzhou, China) to extract total protein from the cultured cells following the manufacturer’s method. SDS‐polyacrylamide gel electrophoresis (10%; SDS‐PAGE) was performed to separate the protein samples (20 µg), which were then transferred to Millipore polyvinylidene difluoride (PVDF) membranes. These PVDF membranes were kept in overnight incubation at 4°C with anti‐CD206, anti‐ODC, anti‐IL‐33, anti‐iNOS, anti‐CD68, anti‐ARG1 and anti‐GADPH primary antibodies (Affinity, USA), followed by incubation at room temperature for 60 minutes with horseradish peroxidase (HRP)–conjugated secondary antibodies (Affinity, USA) that were diluted to 1:5000 in 5% skim milk. After washing with TBST, the membranes were treated for 1 minutes with SuperSignal™ West Dura Extended Duration Substrate (Fudebio, Hangzhou, China) and were visualized through chemiluminescence.
7
Protein Extraction and Western Blot
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Total cellular proteins were extracted with RIPA lysis buffer supplemented with protease and phosphatase inhibitors (Fudebio, Hangzhou, China). The concentration of total proteins was determined with a BCA kit (Thermo, USA). Total proteins (20 µg) were electrophoresed on 10% SDS-PAGE, then electro-transferred to PVDF membrane (Millipore, USA). The membrane was cut into strips and incubated individually with the primary antibodies at 4°C overnight, followed by incubation with the secondary antibodies for 1 h at room temperature. The bands were visualized with enhanced chemiluminescence system (Millipore, USA). The corresponding antibodies are listed in Table S3 .
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