Total proteins were extracted from fresh frozen tissues and cell lines by RIPA lysis buffer. The protein concentration of the supernatant was detected by the BSA Protein Assay Kit (Beyotime institute of Biotechnology, Jiangsu, China). The protein samples were separated on 8% or 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and electrotransferred to nitrocellulose membranes (Millipore, Billerica, MA). After blocking with 5% nonfat milk dilution with TBST (tris-buffered saline with tween-20) for 1 h at room temperature, the membranes were incubated with rabbit anti-RBMS3 antibody (1:1000; Abcam) and rabbit anti-HIF1A antibody (1:1500; Abcam) at 4°C overnight. After washing 3 times with TBST per 10 min, the membranes were then incubated with horseradish peroxidaselabeled anti-rabbit IgG as the secondary antibody (Epitomics) at room temperature for 60 min. Afterwards, after 3 times washing with TBST per 10 min, the membranes were detected with the enhanced chemiluminescence system. Anti-GAPDH antibody (1:3000; vazyme) was used as a loading control.
Bsa protein assay kit
Manufactured by Beyotime
Sourced in China
The BSA protein assay kit is a laboratory equipment used for the quantitative determination of protein concentration. It provides a simple and accurate method to measure the amount of protein present in a sample.
Automatically generated - may contain errors
Lab products found in correlation
Nitrocellulose membrane, by Merck Group (2 mentions)
Ripa buffer, by Beyotime (2 mentions)
Anti gapdh antibody, by Vazyme (1 mentions)
Gsh kit, by Nanjing Jiancheng (1 mentions)
T aoc, by Nanjing Jiancheng (1 mentions)
Icam 1, by Boster Bio (1 mentions)
β actin, by Boster Bio (1 mentions)
X omat film, by Kodak (1 mentions)
Irdye conjugated secondary antibody, by LI COR (1 mentions)
Protease inhibitor cocktail, by Beyotime (1 mentions)
Enhanced chemiluminescence detection reagent, by Thermo Fisher Scientific (1 mentions)
Gadph, by Cell Signaling Technology (1 mentions)
Trpm7 antibody, by Abcam (1 mentions)
Col1a1, by Cell Signaling Technology (1 mentions)
Runx2, by Cell Signaling Technology (1 mentions)
Proteinase inhibitor, by Beyotime (1 mentions)
Enhanced chemi luminescence (ecl), by Beyotime (1 mentions)
Mouse anti β actin, by Merck Group (1 mentions)
6 protocols using bsa protein assay kit
1
Western Blot Analysis of RBMS3 and HIF1A
2
Sophora davidi Fruit Bioactive Analysis
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The fruits of Sophora davidi (Franch.) Skeels were gathered in the south of Qinling Mountains in September 2017 (110°54′ east longitude, 33°32′ north latitude), and authenticated by Prof. Sanqiao Wu from the College of Biological Science and Engineering, Shaanxi University of Technology.
d –galactose, rapid extraction kit of total RNA and ascorbic acid (Vc) were purchased from Sangon Biotech Co., Ltd (Shanghai, China). AChE, iNOS, MDA, SOD, T–AOC and GSH kits were purchased from Jiancheng Bioengineering Institute (Nanjing, China). BSA protein assay kit was purchased from Beyotime Biotechnology (Shanghai, China). DAB reagent kit was purchased from Zhongshan Jinqiao Biotechnology Co., Ltd (Beijing, China). SIRT1 antibody and p53 antibody were acquired from Biosynthesis Biotechnology Co., Ltd. (Beijing, China). cDNA reverse transcription kit and PCR kit were purchased from Takara Biomedical Technology Co., Ltd. (Dalian, China). Hematoxylin, eosin, and immunohistochemistry pen were obtained from Dingguo Changsheng Biotechology Co., Ltd. (Beijing, China).
3
Western Blot Analysis of Retina Proteins
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After isolation of the whole retina from eyeball, RIPA buffer (Beyotime Biotech, China) was applied to retinas for the homogenates (1 hour, on the ice). Protein concentrations were measured using a BSA protein assay kit (Beyotime Biotech). 30 μg protein per sample was separated on 10% SDS-PAGE and then transferred onto nitrocellulose membranes (Millipore). The membranes were blocked in 5% milk or 3% BSA in Tris-buffered saline with 0.5% Tween 20 (TBST) for 1 hour at room temperature on shaking table. The membrane was incubated overnight at 4°C with primary antibodies, including p65 (BIOSS, 1 : 200), p-p65 (BIOSS, 1 : 200), ICAM-1 (Boster, 1 : 400), and β-actin (Boster, 1 : 400). On the next day, after washing with TBST, membranes were incubated with horseradish peroxidase-conjugated secondary antibody (1 : 10000) on the shaking table for 1 hour at room temperature. After washing the membranes with TBST, protein bands were detected by using ECL reagent and obtained images after exposure on X-OMAT film (Kodak). The intensity of the protein band was semiquantitatively measured with image analysis software (ImageJ v2.1, USA), and β-actin was used for calibration.
4
Quantification of SIRT1 and Nrf2 in HUVEC
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Total protein was extracted from treated HUVECs 24 h after treatment with H2O2 and/or SFN or 48 h post-transfection, using a protease inhibitor cocktail added to RIPA buffer (Beyotime Institute of Biotechnology). Total protein was quantified using the BSA Protein Assay kit (Beyotime Institute of Biotechnology) and 30 µg protein/lane was separated by 12% SDS-PAGE. Following electrophoresis, protein samples were transferred onto nitrocellulose membranes and blocked with 5% fat-free milk at room temperature. Membranes were incubated with primary antibodies against SIRT1 (1:1,000; cat. no. ab189494), Nrf2 (1:1,000; cat. no. ab137550) and GAPDH (1:5,000; cat. no. ab9485; all from Abcam) for 12 h at 4˚C. The membranes were subsequently washed and incubated with IRDye®-conjugated secondary antibody (1:2,000; LI-COR Biosciences, cat. no. 926-32211) at room temperature for 2 h. Protein bands were visualized using a Li-Cor Odyssey system v 1.60 (LI-COR Biosciences).
5
Protein Expression Analysis in Bone Cells
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Cells were lysed by sonication with ice-cold lysis buffer containing RIAP protein lysate and a phosphatase inhibitor. A bovine serum albumin (BSA) protein assay kit (Beyotime, Shanghai, China) was used to detect the protein concentrations. The protein sample was separated on a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto a 4.5 μM PVDF membrane (Millipore, Shanghai, China). The membrane was blocked with 5% nonfat milk PBST and then incubated with anti-TRPM7 (1 : 1500), anti-PLC (1 : 1500), anti-P-PLC (1 : 1500), anti-SMAD1 (1 : 1500), anti-P-SMAD1 (1 : 1500), anti-COL1A1 (1 : 1500), anti-ALP (1 : 1500), anti-RUNX2 (1 : 1500), and anti-GADPH (1 : 2000) antibodies at 4°C overnight. Antibodies to PLC, Phospho-PLC, SMAD1, Phospho-SMAD1, RUNX2, COL1A1, and GADPH were purchased from Cell Signaling (Shanghai, China). The TRPM7 antibody was purchased from Abcam (Shanghai, China). After incubation, the membrane was reprobed with the appropriate secondary antibodies (conjugated with horseradish peroxidase) for 1 h. The enhanced chemiluminescence detection reagent (Thermo Scientific, Shanghai, China) and the Tanon 6600 Luminescence Imaging Workstation (Tanon, China) were used to visualize the protein bands. Western blot images were semiquantitatively analyzed with ImageJ software (NIH, USA).
6
Protein Expression Analysis of Liver and Adipose Tissues
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The liver subcutaneous adipose and visceral adipose tissues of every group were homogenized in liquid nitrogen, and whole-cell protein was extracted by using lysate buffer containing proteinase inhibitor (Beyotime Biotechnology, Shanghai, China). Protein concentration was quantified spectrophotometrically by using BSA protein assay kit (Beyotime Biotechnology, Shanghai, China). Protein samples were separated by PAGE using 10% SDS-polyacrylamide gels. Samples were transferred to polyvinylidene fluoride membrane and blocked with 5% milk. The membrane was incubated with a rabbit anti-CDKAL1 primary antibody (1 : 500, Abcam, Cambridge, England) overnight at 4°C and followed by the secondary antibody (against rabbit, Beyotime Biotechnology, Shanghai, China) for 1 h at 37°C. The primary antibodies including mouse anti-β-actin (1 : 1000, Sigma, America) were similar. Lastly, each protein band was detected using enhanced chemiluminescence (ECL, Beyotime Biotechnology, Shanghai, China). The densitometric values were measured with Gel-Pro Analyzer.
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