Cells were lysed by RIPA buffer (Beyotime, Shanghai, China) supplemented with protease inhibitors. Protein concentration was measured using the BCA assay (Beyotime) in accordance with the manufacturer's instructions. Total protein was electrophoresed by SDS-PAGE. The proteins were then transferred to a polyvinylidene fluoride membrane (Millipore, Billerica, MA, USA) and blocked for 1 h with 5% skim milk at room temperature. Incubation with primary antibodies was performed overnight at 4°C. The blots were incubated with horseradish peroxidase (HRP)-labeled secondary antibodies, and the signal was detected using ECL (Beyotime). The following antibodies were used for Western blot: rabbit anti-GAPDH (1:500; Beyotime, Shanghai, China), rabbit anti-CCR5 (1:1000; Protein Tech, Wuhan, China), rabbit anti-caspase 3 (1:1000; Protein Tech, Wuhan, China), rabbit anti-cleaved caspase 3 (1:1000; Cell Signaling Technology, Danvers, Massachusetts, USA), and HRP-labeled goat anti-rabbit secondary antibody (1:5,000; Beyotime, Shanghai, China).
Rabbit anti gapdh
Manufactured by Beyotime
Sourced in United States
Rabbit anti-GAPDH is a primary antibody that specifically binds to the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein. GAPDH is a widely expressed and highly conserved enzyme involved in the glycolytic pathway. This antibody can be used for the detection and quantification of GAPDH in various sample types using techniques such as western blotting, immunohistochemistry, and ELISA.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (2 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Enhanced chemi luminescence (ecl), by Beyotime (1 mentions)
Rabbit anti cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Rabbit anti caspase 3, by Proteintech (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
Bca assay, by Beyotime (1 mentions)
Hrp labeled goat anti rabbit secondary antibody, by Beyotime (1 mentions)
Enhanced chemiluminescence, by Biosharp (1 mentions)
Rabbit anti inos, by Proteintech (1 mentions)
Rabbit anti arg1, by Proteintech (1 mentions)
Bca protein analysis kit, by Thermo Fisher Scientific (1 mentions)
Enhanced chemi luminescence (ecl), by Cytiva (1 mentions)
Enhanced chemiluminescence detection kit, by Thermo Fisher Scientific (1 mentions)
Mouse anti β actin, by Beyotime (1 mentions)
Rabbit anti p16, by Proteintech (1 mentions)
Radioimmunoprecipitation assay (ripa), by Merck Group (1 mentions)
Complete protease inhibitor cocktail, by Merck Group (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Beyotime (1 mentions)
4 protocols using rabbit anti gapdh
1
Western Blot Analysis of Cellular Proteins
2
Western Blot Analysis of Key Proteins
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Levels of iNOS, Arg1, N protein, and GAPDH were measured by Western blotting. Briefly, treated cells were collected and lysed on ice for 30 min in a protein isolation buffer, subjected to SDS-PAGE, and then transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA, USA). The membrane was blocked with 5% low-fat milk at 37 °C for 1 h and then probed with MAb anti-N (1:100; prepared in the College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, China), rabbit anti-iNOS (1:2000, Proteintech), rabbit anti-Arg1 (1:1000; ProteinTech, Rosemont, IL, USA), and rabbit anti-GAPDH (1:1000; Beyotime Biotechnology, Shanghai, China) at 37 °C for 2 h. After washing, the membrane was incubated with HRP-conjugated anti-mouse or anti-rabbit secondary antibody (1:10,000, ABclonal). Bound proteins were detected by enhanced chemiluminescence (Biosharp, Hefei, China) and imaged using a chemiluminescence imaging system (Tanon, Shanghai, China).
3
Hippocampal Protein Extraction and Western Blot
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Hippocampal tissue removed from storage at − 80°C was dissolved with RIPA lysis buffer containing a mixture of protease inhibitors and phosphatase inhibitors. The solution was centrifuged at 12000 g for 10 min and then supernatant were analyzed. Protein concentration was determined using the BCA Protein analysis kit (Thermo Fisher Scientific, Inc.). A total of 25 μg of proteins were isolated using 10% or 12% SDS polyacrylamide gel and transferred to PVDF membrane. After sealing with 5% skim milk for 1 h, the membrane was incubated with primary antibodies overnight on a shaker screen. Primary antibodies contained rabbit anti-xCT (1:500 dilution, Boster, BM5318), rabbit anti-Gpx4 (1:1000 dilution, Proteintech, Cat No: 67763-1-Ig) and rabbit anti-PSD-95 (1:2000 dilution, Abcam, ab238135). Rabbit-anti-GAPDH (1:1000, Beyotime, AF0006) for load control. After washing in Trisbufered brine, the membranes were incubated with corresponding secondary antibodies (diluted at 1:5000, Abcam, Cambridge, UK). Cross reactivity was observed using western blotting assay of ECL (Amersham Pharmacia Biotech, Piscataway, NJ, USA), and then Lab imaging system was analyzed by scanning optical density analysis.
4
Western Blot Analysis of Lung Proteins
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The RIPA (Sigma-Aldrich) buffer containing complete Protease Inhibitor Cocktail (Sigma-Aldrich) and PMSF (Beyotime, China) was used to extract proteins from lung tissues and cells. The extracts were centrifuged at 4℃ for 15 min at 12,000 rpm and the supernatants were quantified with the BCA protein assay kit (Beyotime). Equal amounts of total protein were separated by SDS-PAGE and transferred to PVDF membranes (Millipore, USA). Membranes were blocked with 5% skim milk at room temperature for 1 h and then incubated with the primary antibodies at 4℃ overnight. After washing with TBST (Tris-buffered saline, 0.1% Tween-20), the membranes were subsequently incubated with secondary antibodies for 1 h at room temperature. Protein bands were visualized with the Enhanced Chemiluminescence Detection Kit (Thermo Fisher) via the Tannon luminescent imaging system. Image J was used for the quantitative analysis of the protein bands. The antibodies used are listed below: rabbit-anti-Bmi-1(CST, 1:2000 dilution), rabbit-anti-p16 (Proteintech, USA, 1:1000), mouse-anti-β-actin (CST, 1:5000 dilution), rabbit-anti-GAPDH (CST, 1:5000 dilution), rabbit-anti-pH2A.X (Beyotime, 1:1000).[33 (link)].
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