Cells lysates were prepared using RIPA buffer (Beyotime, China) containing PMSF and phosphatase inhibitor (Servicebio, China). The extracts were clarified by centrifugation at 12,000 rpm for 10 min at 4°C and total protein concentrations estimated using the BCA Protein Assay Kit (Beyotime, China). Equal protein amounts were prepared in SDS-PAGE loading buffer and heated at 100°C for 5 min before protein separation by SDS PAGE and transfer to PVDF membranes (Millipore). After blocking with skim milk solution for 90 min, membranes were incubated with the indicated primary antibodies. (Src rabbit monoclonal antibody, 1:1000, CST; Phospho-Src Family (Tyr416) (D49G4) rabbit monoclonal antibody, 1; 1000, CST; t-FUNDC1 rabbit polyclonal antibody, 1:500, abcepta; p-FUNDC1 (Tyr18) rabbit polyclonal antibody, 1:500, abcepta; LC3 rabbit polyclonal antibody, 1:1000, Proteintech; P62 rabbit polyclonal antibody, 1:1000, CST; GAPDH Mouse monoclonal antibody, 1:3000, Antgene, China; Detailed antibody information is provided in Supplementary Table S1 ) followed by the appropriate anti-rabbit or mouse HRP-conjugated secondary antibodies. The membranes were incubated with ECL solution and protein bands visualized in an imaging system. The intensities of bands were quantified using Image J software. Uncropped Western blot images are provided in Supplementary Figure S1 .
Gapdh mouse monoclonal antibody
Manufactured by Antgene
Sourced in China
The GAPDH Mouse monoclonal antibody is a laboratory reagent designed for the detection and quantification of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein in biological samples. GAPDH is a key enzyme involved in the glycolytic pathway and is widely used as a housekeeping gene for normalization in various analytical techniques.
Automatically generated - may contain errors
Lab products found in correlation
Ripa buffer, by Beyotime (2 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Phosphatase inhibitor, by Wuhan Servicebio Technology (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Wuhan Servicebio Technology (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Lc3 rabbit polyclonal antibody, by Proteintech (1 mentions)
Nitrocellulose membrane, by GE Healthcare (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Odyssey infrared imaging system, by LI COR (1 mentions)
Alexa fluor 680 790 labeled goat anti rabbit goat anti mouse igg antibody, by LI COR (1 mentions)
2 protocols using gapdh mouse monoclonal antibody
1
Western Blot Analysis of Cellular Signaling
2
Protein Extraction and Western Blot Analysis in Glomeruli and Podocytes
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Total protein from the glomeruli and podocytes was extracted with RIPA buffer (Beyotime, China) mixed with a protease inhibitor cocktail (Sigma- Aldrich, USA). The extractions were then centrifuged at 13,000 rpm for 10 min at 4°C. The supernatants were mixed with loading buffer prior to being boiled at 100°C for 5 min. Equal amounts of protein samples were separated through SDS-PAGE and then transferred to nitrocellulose membranes (GE Healthcare). Membranes were blocked with milk for 1-2 h before incubation with a primary antibody (Sirt6 rabbit monoclonal antibody, 1:1000, Abcam; p-AMPKα1/2 (Thr172) rabbit polyclonal antibody, 1:100, Santa Cruz Biotechnology; AMPKα1/2 (H-300) rabbit polyclonal antibody, 1:100, Santa Cruz Biotechnology; caspase-3 mouse monoclonal antibody, 1:1000, Novus Biologicals; GAPDH rabbit monoclonal antibody, 1: 1,000, Antgene and GAPDH mouse monoclonal antibody, 1: 1,000, Antgene) overnight at 4°C. An Alexa Fluor 680/ 790-labeled goat anti-rabbit/goat anti-mouse IgG antibody (1:10,000, LI-COR Biosciences, USA) was used as the secondary antibody, and the blots were visualized using a LI-COR Odyssey Infrared Imaging System.
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