Total proteins were separated with radioimmunoprecipitation assay buffer (Biosharp, Hefei, China) containing 1 mM phenylmethylsulfonyl fluoride and a phosphatase inhibitor cocktail (MedChemExpress, NJ, USA). After centrifugation at 12,000 rpm for 15 min at 4°C, the concentration of proteins was determined with the BCA protein assay kit (AMEKO, Shanghai, China). The proteins (30 μg) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis using 10% gels (Epizyme, Shanghai, China) and transferred to polyvinylidene difluoride membranes. We cut the membranes horizontally into strips for detecting proteins with different molecular weights. The membranes were blocked with blocking buffer (Biosharp) for 1–4 h and incubated with primary antibody for 12 h at 4°C. Next, the membranes were incubated with secondary antibody for 1 h at room temperature, followed by the detection of target proteins with an ultra-sensitive ECL chemiluminescence kit (NCM Biotech, Suzhou, China) and the ImageQuant LAS 500 system (GE Healthcare, Fairfield, CT, USA). The intensity of each band was quantified by ImageJ software (Bethesda, MD, USA) and normalized using β-actin.
Radioimmunoprecipitation assay buffer
Manufactured by Biosharp
Sourced in China
Radioimmunoprecipitation assay buffer is a laboratory reagent used in the process of immunoprecipitation. It is designed to facilitate the extraction and purification of protein complexes from cell or tissue samples.
Automatically generated - may contain errors
Lab products found in correlation
Phosphatase inhibitor cocktail, by MedChemExpress (1 mentions)
Blocking buffer, by Biosharp (1 mentions)
Imagequant las 500 system, by GE Healthcare (1 mentions)
Phosphatase inhibitors, by Biosharp (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Gadph, by Proteintech (1 mentions)
Bca protein quantitation kit, by Thermo Fisher Scientific (1 mentions)
Hrp conjugated secondary antibody, by Proteintech (1 mentions)
Bicinchoninic acid method, by Thermo Fisher Scientific (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Ecl reagent, by Abcam (1 mentions)
Electrochemiluminescence detection system, by Thermo Fisher Scientific (1 mentions)
Bicinchoninic acid protein assay kit, by Biosharp (1 mentions)
β actin 3700, by Cell Signaling Technology (1 mentions)
Vegfa 66 828 1 ig, by Proteintech (1 mentions)
Image lab 5, by Bio-Rad (1 mentions)
5 protocols using radioimmunoprecipitation assay buffer
1
Quantitative Protein Analysis by Western Blot
2
Western Blot Analysis of Leptin
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Fresh samples were extracted and homogenized with radioimmunoprecipitation assay buffer (Biosharp, Hefei, China) on ice containing phenylmethanesulfonyl fluoride (Biosharp) and phosphatase inhibitors (Roche, Basel, Switzerland). The protein concentration was quantified with the BCA Protein Quantitation Kit (Thermo Scientific). The proteins were denatured at 95 °C for 5 min and diluted with 5 × loading buffer. The protein samples were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride membrane (Millipore, Paisley, UK). The membranes were blocked with 5% BSA at room temperature for 2 h and then incubated with primary leptin antibody (1:1000; Abcam, Cambridge, UK). The membranes were washed in Tris-buffered saline containing 0.1% Tween-20 and incubated with an HRP-conjugated secondary antibody for 2 h (1:5000) (Proteintech, Rocky Hill, NJ, USA) at room temperature. The band densities were analyzed by ImageJ software and GADPH (1:5000; Proteintech) was used as the control.
3
Western Blot Analysis of Protein Extracts
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Total proteins were extracted from RTECs using a radioimmunoprecipitation assay buffer (Biosharp) and quantified by the bicinchoninic acid method (Thermo Fisher Scientific). After denaturing, proteins were separated using 12% sodium dodecyl sulphate polyacrylamide gel electrophoresis. Next, the gel was transferred to polyvinylidene difluoride membranes (Millipore), and membranes were blocked in 5% fat‐free milk for 2 h at room temperature. Membranes were then incubated with specific antibodies at 4°C overnight followed by HRP‐conjugated anti‐rabbit secondary antibody for 1 h at room temperature. Blots were developed using the ECL reagent (Abcam). The gray values were analyzed in Image J v1.46 software (National Institutes of Health). Used antibodies are listed in Table 1 .
4
Western Blot Analysis of TIMP1
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Radioimmunoprecipitation assay buffer (Biosharp, China) was used to extract the total protein from the cells and CRC tissues. Proteins were separated using 8%–12% SDS–PAGE and transferred onto polyvinylidene difluoride membranes. The membranes were blocked with quick‐block fluid and incubated with primary antibodies (Bioss, China), including TIMP1, overnight at 4°C. The bands were measured using an enhanced chemiluminescence substrate after being incubated for 2 h at room temperature with secondary antibodies.
5
Western Blot Analysis of Endothelial Cells
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HUVECs and BMECs were lysed using radioimmunoprecipitation assay buffer (Biosharp, Hefei, China). To determine protein concentration, a Bicinchoninic Acid Protein Assay Kit (Biosharp) was utilized. Equivalent protein lysates were separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes. Then they were then subjected to western blot analysis. The primary antibodies against FOXO1 (2880S), cleaved caspase-3 (9661S), and β-actin (3700S) were purchased from Cell Signaling Technology (Danvers, MA, USA). The primary antibodies anti-cyclin D1 (60186-1-1), anti-cyclin E1 (11554-1-AP), anti-Bim (22037-1-AP), anti-Bcl2 (60178-1-Ig), anti-Bax (60267-1-Ig), anti-matrix metalloproteinase (MMP)2 (66366-1-Ig), anti-MMP9 (N-terminal, 10375-2-AP), and anti-vascular endothelial growth factor A (VEGFA; 66828-1-Ig) were obtained from Proteintech. In addition, horseradish peroxidase-conjugated goat anti-mouse or anti-rabbit IgG H&L (ZJ2020-M and ZJ2020-R, respectively; Bioworld) was applied as the secondary antibody. The protein bands were visualized via an electrochemiluminescence detection system (Thermo Fisher Scientific, Waltham, MA, USA) and analyzed using Image Lab 5.0 software (Bio-Rad, Hercules, CA, USA).
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