Proteins from samples were isolated using RIPA buffer (Vazyme, Nanjing, China) and were segregated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, and then proteins were transferred onto the polyvinylidene difluoride membranes (Vazyme). The membranes were blocked with 5% skimmed milk (Vazyme). Thereafter, the membranes were incubated with the primary antibodies: anti-Irak2 (1:2000, ab62419, Abcam, Cambridge, UK) or glyceraldehyde 3-phosphate dehydrogenase (1:2500, ab9485, Abcam) overnight. After being rewashed, the membranes were incubated with the secondary antibody (1:3000, ab205718, Abcam) for 2 h. The membranes were analyzed by the ChemiDoc™ MP Imaging System (Bio-Rad) after being treated with ECL kit (Vazyme).
Ripa buffer
Manufactured by Vazyme
Sourced in China
RIPA buffer is a commonly used protein extraction and lysis buffer. It is designed to efficiently lyse cells and solubilize proteins, while maintaining their native structure and interactions.
Automatically generated - may contain errors
Lab products found in correlation
Ab205718, by Abcam (5 mentions)
Chemidoc mp imaging system, by Bio-Rad (5 mentions)
Ecl kit, by Vazyme (4 mentions)
Pvdf membrane, by Merck Group (4 mentions)
Pvdf membranes, by Vazyme (3 mentions)
Detergent compatible bradford protein quantification kit, by Vazyme (3 mentions)
Protease inhibitor cocktail, by Roche (3 mentions)
Bca kit, by Thermo Fisher Scientific (3 mentions)
Ab9485, by Abcam (2 mentions)
Enhanced chemiluminescence, by Vazyme (2 mentions)
G box chemi xx9 imaging system, by Syngene (2 mentions)
Gapdh, by Cell Signaling Technology (2 mentions)
Horseradish peroxidase, by Proteintech (2 mentions)
Ab40772, by Abcam (2 mentions)
Ab8227, by Abcam (2 mentions)
Pvdf membrane, by Thermo Fisher Scientific (2 mentions)
Pvdf membrane, by Bio-Rad (2 mentions)
Pvdf membrane, by Roche (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Bca protein assay, by Thermo Fisher Scientific (2 mentions)
24 protocols using ripa buffer
1
Western Blot Protein Detection
2
Quantitative Protein Analysis of HepG2 Cells
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Total proteins from transfected and untransfected HepG2 cells were extracted using RIPA buffer (Vazyme Biotech, China). BCA assay (Beyotime, China) was conducted to quantify the extracted total proteins. For isolation of the protein samples, 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was carried out followed by transfer to polyvinylidene fluoride membranes (0.45 μm) with 300 mA current for 90 min. After transfer, the membranes were blocked in 5% BSA at ambient temperature for 1 h and then incubated overnight using rabbit antibodies against GPC3 (Abcam, USA; diluted 1 : 1000) and a rabbit antibody against GAPDH (Sangon Biotech, China; diluted 1 : 5000) as an internal standard at 4°C to normalize the protein expressions. On the next day, the membranes were further incubated using goat anti-rabbit IgG-HRP (Sangon Biotech, China; diluted 1 : 5000) for 1 h and then subjected to protein visualization using enhanced chemiluminescence (Vazyme Biotech, China). The G:BOX Chemi XX9 imaging system (Syngene, UK) was used for protein visualization.
3
Protein Quantification and Western Blot Analysis
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Proteins were isolated by RIPA buffer (Vazyme), and Detergent Compatible Bradford Protein Quantification Kit (Vazyme) was used to determine the concentration of proteins. Subsequently, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to separate the proteins and then the proteins were transferred onto the polyvinylidene difluoride (PVDF) membranes (Vazyme). After being blocked with 5% skimmed milk (Vazyme) and washed by phosphate-buffered saline (PBS), the membranes were incubated with corresponding primary antibodies: MUC19 (1:1000 dilution, ab212621, Abcam, Cambridge, UK), GAPDH (1:2000, ab37168, Abcam), PCNA (1:3000, Abways Technology, Inc., Shanghai, China), cyclin D1 (1:1000, Abways Technology), and antibodies against hexokinase II (HK2) (ab227198, 1:5000, 102 kDa) overnight at 4 °C. Then the membranes were incubated with the secondary antibody (1:3000, ab205718, Abcam) for 3 h. The blots were analyzed by the ChemiDoc™ MP Imaging System (Bio-Rad, Richmond, CA, USA) after being incubated with ECL kit (Vazyme).
4
Western Blot Analysis of Key Signaling Proteins
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Samples were harvested with RIPA buffer (Vazyme) and processed with a lysis buffer containing phenylmethanesulfonyl fluoride (PMSF; Amresco) at 4°C. BCA protein assay kit (Beyotime Institute of Biotechnology, China) was used to measure protein concentration according to the manufacturer's protocol. Proteins were separated by 12% SDS-PAGE gels, then transferred to PVDF membranes (Bio-Rad, Calif) and blocked in 5% nonfat milk for 2 h. Subsequently the membranes were botted with specific primary antibodies against Ser9phospho GSK-3β (Ser9; 1:1,000, Cell Signaling), total GSK-3β (1:1,000, Cell Signaling), cleaved caspase-3 (1:1,000, Cell Signaling), HDAC3 (1:1,000, Cell Signaling), and GAPDH (1:1,000, Abcam) overnight at 4°C. The next day, membranes were washed 3 times with PBS-T for 10 min followed by incubation with the corresponding HRP-conjugated secondary antibodies (1:8,000, Abcam) for 1 h at 37°C. Membranes were then washed with PBS-T 3 times. The bands were visualized with enhanced ECL reagent (Thermo Scientific) and analyzed using Quantity One software. GAPDH was used as a control.
5
Western Blot Analysis of Cellular Proteins
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Cells were lysed using the RIPA buffer (Vazyme). Lysates (30 μg/sample) were loaded on 10% denaturing SDS-PAGE gels and transferred onto PVDF membranes (Roche, Basel, Switzerland). The latter were blocked for 2 h in Tris-HCl buffer containing 5% bovine serum albumin, followed by incubation with primary antibodies overnight at 4°C. The membranes were then labeled with secondary antibodies at 28°C for 1 h. The antibodies used included those against FAF1 (Cat: 10271-1-AP, Proteintech, Chicago, IL, USA), β-catenin (Cat: 51067-2-AP, Proteintech), and GAPDH (Cat: 5174, Cell Signaling, Boston, MA, USA), and the anti-rabbit IgG, HRP-linked antibody (Cat: 7074, Cell Signaling).
6
Western Blot Analysis of Epigenetic Regulators
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The cells were washed in PBS and lysed with RIPA buffer (Vazyme, China) supplemented with protease inhibitor cocktail (Roche, Germany). After quantification using One DropTM 1000+ (ANTPEDIA, China), proteins were separated by SDS-PAGE under denaturing conditions and transferred to PVDF membranes (Millipore, USA). Membranes were blocked with 5% fat-free milk for 2 h at room temperature and incubated with primary antibodies for Gapdh (Proteintech, 60004-1-Ig, 1:5,000), Mll1 (CST, D668N, 1:1,000), Mll2 (CST, E6A8V, 1:1,000), Pax6 (Abcam, ab5790, 1:500), and H2b (Abcam, ab52599, 1:10,000). The membranes were then incubated with secondary antibodies conjugated with horseradish peroxidase (Proteintech, USA). Protein signals were visualized using the ECL detection reagent (Thermo Scientific) on UVP (Analytik Jena AG, Germany).
7
Western Blot Protein Quantification and Analysis
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As previously described [22 (link)], cells were washed twice with PBS and solubilized with radio immunoprecipitation assay (RIPA) buffer (Vazyme Biotec Co., LTD, Nanjing, China) supplemented with protease inhibitor (Sigma). Lysates were quantitated with bicinchoninic acid (BCA) kit (Vazyme Biotec Co., LTD); equivalent amounts of protein were subjected to electrophoresis, and transferred onto PVDF membranes (Millipore, Billerica, MA, USA). Blocking with 5% dried skimmed milk in TBST (Tris Buffered Saline and 0.1% Tween 20) for 2 h at room temperature, the membranes were incubated with different primary antibodies, including KLF13 (#41724, SAB, Maryland, MD, USA), FASN (sc-20140 AC, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), FABP4 (sc-18661, Santa Cruz Biotechnology, Inc.), PPARγ (#2435, CST, Cell Signaling Technology, Inc., Danvers, MA, USA), and β-tubulin (KM9003, Sungene, Tianjin, China), at 4 °C overnight. After washing, the membrane was incubated with HRP-conjugated secondary antibodies for 1 h at room temperature and developed with ChemiDocTM XRS + Chem iluminescence detection system (Bio-Rad, Hercules, CA, USA). Image Lab5.2 was used for densitometric analysis of the expressed protein bands.
8
Western Blot Analysis of Autophagy Markers
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Western blot analysis was performed to detect the protein expression levels of SPRED2, microtubule-associated protein light chain 3 (LC3)-II, LC3-I and Beclin 1. Briefly, treated cell lines were harvested and washed with PBS. Total protein was extracted from cells using RIPA buffer (Vazyme Biotech Co., Ltd.). Protein was then quantified using the BCA™ Protein Assay kit (Merck KGaA). Samples (30 µg) were then separated by 10% SDS-PAGE (Thermo Fisher Scientific, Inc.). The separated proteins were subsequently transferred onto PVDF membranes (Amersham; Cytiva) and blocked with 5% non-fat milk at room temperature for 2 h. The membranes were incubated with primary antibodies against thrombin (cat. no. ab92621; 1:1,000), SPRED2 (cat. no. ab153700; 1:500), Beclin 1 (cat. no. ab210498; 1:1,000), LC3-II/I (cat. no. ab62721; 1:2,000) and GAPDH (cat. no. ab181602; 1:10,000) overnight at 4˚C (all purchased from Abcam). Following the primary incubation, membranes were incubated with a horseradish peroxidase-conjugated secondary antibody (cat. no. ab205718; 1:2,000; Abcam) at 37˚C for 45 min. Protein bands were visualized by Pierce ECL Western Blotting Substrate (Pierce; Thermo Fisher Scientific, Inc.) using the Bio-Image Analysis System (Bio-Rad Laboratories, Inc.). GAPDH served as the internal control.
9
Western Blot Analysis of Protein Signaling
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After lysing in radioimmunoprecipitation assay (RIPA) buffer (Vazyme, P.R. China), which contained protease inhibitors (Roche, Germany), cells were washed with D-Hanks solution. Proteins were quantified with the bicinchoninic acid (BCA) protein assay kit (Vazyme, P.R. China). Samples containing 30 μg of total proteins were electrophoresed on SDS-polyacrylamide gels and then transferred onto a polyvinylidene fluoride (PVDF) membrane (Millipore, USA) by electroblotting using a Bio-Rad Bis-Tris gel system (Bio-Rad, USA). The membranes that were covered with the primary antibody were blocked by 5% nonfat milk and incubated overnight at 4°C, then washed with Tris-buffered saline with Tween 20 (TBST), and subsequently incubated with a secondary antibody for 1 h at 37°C. Finally, an enhanced chemiluminescence (ECL) solution (Millipore, USA) was added to cover the blotting surface. The signals were captured, and the intensity of the bands was quantified by using the Bio-Rad ChemiDoc XRS+ system (Bio-Rad, USA). Proteins isolated from tissues and cells were incubated with a primary antibody that included MYB (Abcam, USA), GAPDH (SAB, USA), caspase-3, AKT, phosphorylated (p-)AKT (Ser473), PI3K (Cell Signaling Technology, MA, USA).
10
Western Blot Analysis of USP32 Protein
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U-251 MG and U-87 MG cells were lysed using RIPA buffer (Vazyme). The protein concentrations of samples were measured using a bicinchoninic acid (BCA) protein quantification kit (Abcam, Shanghai, China). Samples (12 μg/lane) were loaded into a 12% SDS-PAGE gel for electrophoresis and then transferred onto a PVDF membrane (Roche, Basel, Switzerland). The membrane was incubated with primary antibody Anti-USP32 (1:1000, CAT#ab251903, Abcam) or Anti-GAPDH (1:2000, CAT#ab9485, Abcam) at 4 °C overnight, followed by incubation with secondary antibody HRP-conjugated Goat Anti-Rabbit IgG H&L (1:2000, CAT#ab6721, Abcam) at 28 °C for 30 min. The signals were visualized using the ECL detection system (Thermo Fisher Scientific, Waltham, MA, USA) and quantified by densitometry using Image J v1.48u.
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