Protein samples from femoral head tissues were obtained using a Total Protein Extraction Kit (BC3711, Solarbio). Then, the protein concentration was determined by a Protein Assay kit (Beyotime, Shanghai, China). Protein samples were separated by 10% SDS-PAGE gel and electrically transferred to PVDF membranes (Millipore, Billerica, MA, USA). The membranes were hatched with the primary antibodies at 4℃ overnight after being blocked with 3% bovine serum albumin (BSA, Sangon Biotech, Shanghai, China) for 1 h at room temperature. The primary antibodies were listed as followed: rabbit anti-AKT (ab179463, 1：10,000); rabbit anti-p-AKT (ab81283, 1：1,000); rabbit anti-mTOR (ab2732; 1：10,000); rabbit anti-p-mTOR (ab63552; 1：1,000); rabbit anti-β-actin (ab8227; 1：1,000) (Abcam, Cambridge, UK). The membranes were incubated with goat-anti-rabbit IgG (H＋L)-HRP (1：10,000, ab6721, Abcam) for 1 h at room temperature after washing with TBST thrice. Protein bands were visualized with an Electrochemilluminescence (ECL) chemilumine-scence kit (WBULS0500; EMD Millipore) and the bands intensity were analyzed with Image-Pro Plus 6.0 software.
Ab63552
Manufactured by Abcam
Sourced in United Kingdom, United States
Ab63552 is a recombinant monoclonal antibody specific for the protein of interest. It is produced in mammalian cells and purified by affinity chromatography.
Automatically generated - may contain errors
Lab products found in correlation
Ab6721, by Abcam (1 mentions)
Ab2732, by Abcam (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Protein assay kit, by Beyotime (1 mentions)
Wbuls0500, by Merck Group (1 mentions)
Ab179463, by Abcam (1 mentions)
Ab8227, by Abcam (1 mentions)
Total protein extraction kit, by Solarbio (1 mentions)
Ab81283, by Abcam (1 mentions)
Ab62557, by Abcam (1 mentions)
Ab109012, by Abcam (1 mentions)
Ab150113, by Abcam (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Nitrocellulose membrane, by Merck Group (1 mentions)
Ab38449, by Abcam (1 mentions)
Ab128025, by Abcam (1 mentions)
Ab189403, by Abcam (1 mentions)
Ab6566, by Abcam (1 mentions)
Ab109268, by Abcam (1 mentions)
Ab32575, by Abcam (1 mentions)
2 protocols using ab63552
1
Protein Extraction and Western Blot Analysis
2
Protein Expression Analysis in NRK-52E Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
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NRK-52E cells were lysed in radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime, Suzhou, China) to extract the total protein. The protein was separated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto nitrocellulose membranes (Sigma-Aldrich, St. Louis, USA). Then, the membranes were placed in 5% milk for 2 h, incubated with 1000 times diluted antibodies of Col IV (ab6566), α-smooth muscle actin (α-SMA, ab32575), p62 (ab109012), microtubule-associated protein 1A/1B-light chain 3 (LC3)-II/I (ab128025), Beclin 1 (ab62557), phosphatase and tensin homolog deleted on chromosome ten (PTEN, ab32199), phosphatidylinositol 3-kinase (PI3K, ab189403), phosphorylated (p)-PI3K (ab182651), total (t)-protein kinase B (t-AKT, ab8805), p-AKT (ab38449), p-mTOR (p-mTOR, ab109268), and t-mTOR (ab63552, Abcam, Cambridge, USA) at 4°C overnight; then anti-mouse immunoglobulin G (IgG) antibody (1:2000; ab150113, Abcam, Cambridge, USA) was added and the membranes were incubated for 1 h. The bands were analyzed by an imaging system (Bio-Rad, Hercules, USA) and ImageJ software (NIH Image, Bethesda, USA).
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