The tissues and cells of total proteins were harvested and lysed with high efficiency RIPA lysate (Solarbio, Beijing, China). The contents of proteins were analyzed using protein analyzer (Imagin 200, MD Pacific, Tianjing, China). Each protein was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The protein was bound to PVDF membranes (Tengxiang; Wenzhou, Zhejiang, China). Then, membranes were transferred to TBST containing 5% skimmed milk powder for 1.5 hours. The corresponding primary antibody was used to incubate the membranes at 4°C refrigerator for 24 hours. (anti-MTHFD1, Abcam, ab18708, dilution: 1: 1000; anti-p53, R&D, IC13551G, dilution: 1: 800; anti-survivin, R&D, IC886G, dilution: 1: 500; anti-Bcl-2 associated X protein (Bax), Abcam, ab32503, dilution: 1: 1000; anti-B-cell lymphoma-2 (Bcl-2), Abcam, ab32370, dilution: 1: 800; anti-DNA methyltransferase 1 (DNMT1), CST, 5032, dilution: 1: 800; anti-DNMT3a, CST, 2160, dilution: 1: 700; anti-DNMT3b, CST, 67259, dilution: 1: 800; anti-GAPDH, R&D, 2275-PC-100, dilution: 1: 1000). Subsequently, membranes were incubated with the secondary antibodies (donkey anti-rabbit IgG, R&D, NL004, 1: 5000; mouse anti-rabbit IgG, CST, 93702, 1: 6000; goat anti-mouse IgG, Abcam, ab6785, 1: 8000) at 37°C for 1 hour. The membranes were exposed by Bio-RAD GelDoc XR+ (V140130, Vedeng, Suzhou, China).
High efficiency ripa lysate
Manufactured by Solarbio
Sourced in China
High-efficiency RIPA lysate is a buffer solution used for the extraction and lysis of protein samples from cells and tissues. It is designed to effectively solubilize proteins while preserving their native structure and function.
Automatically generated - may contain errors
Lab products found in correlation
Ab32503, by Abcam (1 mentions)
Ab32370, by Abcam (1 mentions)
Nl004, by R&D Systems (1 mentions)
Ab6785, by Abcam (1 mentions)
Nuclear protein extraction kit, by BestBio (1 mentions)
Bca kit, by GenStar (1 mentions)
G box chemi xrq, by Syngene (1 mentions)
Ecl chemiluminescence substrate, by Biosharp (1 mentions)
Bs 1428r, by Bioss Antibodies (1 mentions)
Las4000, by GE Healthcare (1 mentions)
Sa00001 2, by Proteintech (1 mentions)
Total protein assay kit, by Nanjing Jiancheng (1 mentions)
Vascular endothelial growth factor (vegf), by Proteintech (1 mentions)
Ecl western blotting substrate, by Solarbio (1 mentions)
Horseradish peroxidase hrp conjugated secondary antibody, by Solarbio (1 mentions)
Pvdf membrane, by Solarbio (1 mentions)
Bca protein assay kit, by Solarbio (1 mentions)
Gapdh, by Proteintech (1 mentions)
4 protocols using high efficiency ripa lysate
1
Western Blot Analysis of Protein Expression
2
Western Blotting Protein Extraction
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The GMECs and mammary tissues were lysed using high-efficiency RIPA lysate (Solarbio, Co., Ltd., Beijing, China) or a nuclear protein extraction kit (BestBio, Co., Ltd., Nanjing, China) to obtain total proteins, nuclear proteins and cytoplasmic proteins. The concentration of protein samples was determined using a BCA kit (Genstar). For Western blotting, samples with a loading buffer (CWBIO, Co., Ltd., Beijing, China) were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis on gels (Jingcai Biological, Co., Ltd., Xi’an, China) and blocked in Tris-buffered saline Tween (TBST) with 5% non-fat milk (BBI, Co., Ltd., Shanghai, China) for 2 h at 25 °C after being transferred to a polyvinylidene fluoride membrane (Millipore, Sigma-Aldrich). The membranes were then incubated with specific primary antibodies at 4 °C overnight and with horseradish peroxidase-conjugated secondary antibodies (1:4000, diluted in TBST) at 25 °C for 2 h. The dilution ratios of the primary antibodies are listed in Table A2 . After antibody incubation, the membrane was washed with TBST and photographed using a G:BOX Chemi XRQ (Syngene, Co., Frederick, MD, USA) imaging system.
3
Quantifying Tight Junction Proteins in Mice
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The samples were prepared by grinding the colonic tissue of experimental mice with high-efficiency RIPA lysate (Solarbio, Beijing, China) supplemented with protease inhibitors. A total protein assay kit (Jiancheng, Nanjing, China) was used to determine the protein content of the samples. The samples were separated by polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane. The membrane was sealed with 5% skim milk powder for 1 h at room temperature. After incubation, proteins on the membrane were detected with ECL chemiluminescence substrate (Biosharp, HeFei, China) and blotted on a gel imaging system (LAS4000, GE, Boston, MA, USA). Primary antibodies against Claudin-1 (Bioss, Woburn, MA, USA, bs-1428R, 1/1000), Occludin (Bioss, bs-10011R, 1/1000), SOCS1 (Abcepta, San Diego, CA, USA, AP8790A, 1/1000), β-actin (Proteintech, Rosemont, IL, USA 20536-1-AP, 1/2000) and secondary antibody (Proteintech, SA00001-2, 1/5000) were used for Western blot analysis. The intensity of the protein bands was analyzed using image analysis software (Image J 1.53a, Bethesda, MD, USA).
4
Protein Extraction and Western Blot Analysis
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To obtain total protein from cells, high-efficiency RIPA lysate (Cat no. R0010; Solarbio, Beijing, China) was used. Then, the speed of the high-speed centrifuge was set at 16,000 × g, the temperature was set at 4°C, and the centrifugation was performed for a total of 15 min. The protein concentration was then quantified by BCA protein assay kit (Cat no. PC0020, Solarbio, Beijing, China). The protein was denatured after being treated at 95°C for 5 min, followed by SDS-PAGE (7.5%), and the loading amount of protein per pore was 30 μg. After electrophoresis, the protein was transferred to PVDF membrane (Solarbio, Beijing, China) by wet transfer method, blocked with 5% skimmed milk dissolved in TBST prepared in advance, and placed on a shaker for 2 h at room temperature. The corresponding primary antibodies were used to incubate the membranes.
The primary antibodies were as follows: VEGF (Cat no. 19003-1-AP; Proteintech, Shanghai, China) and GAPDH (1 : 2,000) (Cat no. 60004-1-Ig; Proteintech, Shanghai, China). Then, the membranes were incubated with the horseradish peroxidase (HRP)-conjugated secondary antibody (1 : 2000) (Solarbio, Beijing, China) for 2 h at 37°C. ECL Western Blotting Substrate (Solarbio, Beijing, China) was used to detect the intensity of chemiluminescence. Image J version 1.53 was used to analyze results.
The primary antibodies were as follows: VEGF (Cat no. 19003-1-AP; Proteintech, Shanghai, China) and GAPDH (1 : 2,000) (Cat no. 60004-1-Ig; Proteintech, Shanghai, China). Then, the membranes were incubated with the horseradish peroxidase (HRP)-conjugated secondary antibody (1 : 2000) (Solarbio, Beijing, China) for 2 h at 37°C. ECL Western Blotting Substrate (Solarbio, Beijing, China) was used to detect the intensity of chemiluminescence. Image J version 1.53 was used to analyze results.
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