Approximately 1× 106 PBMCs were treated with cell lysis buffer (Cell Signaling Technology) from a subset of 5 young responders, 5 young non-responders, 5 older responders, and 5 older non-responders for which sufficient cell amounts were available. Lysed samples were briefly sonicated, and kept on ice for an additional 10 minutes. Lysates were mixed with 2x Laemmli Sample buffer, boiled and loaded onto 4-20% mini protean ready gels (BioRad). Following transfer, membranes were blocked with 5% milk powder in 1X TBS-tween 20 buffer at room temperature for 1 hour. Blocked membranes were incubated with primary antibodies against human HSP60 (Cell Signaling Technology), Succinate Dehydrogenase (SDHA) (Cell Signaling Technology) and HRP conjugated beta actin (anti-human beta-actin, Sigma), following by an HRP conjugated anti-Rabbit secondary antibody (Cell Signaling Technology) in 1x TBS-tween 20 containing 5% milk powder. Membranes were washed and developed and pixel intensities of the specific protein band and corresponding actin band on autoradiographs were estimated using ImageJ software. The relative pixel intensities (signal/actin) were plotted and statistical significance evaluated using a t-test.
Anti human beta actin
Manufactured by Merck Group
Anti-human beta-actin is a monoclonal antibody used in laboratory applications to detect and quantify the expression of the beta-actin protein. Beta-actin is a widely expressed cytoskeletal protein that plays a fundamental role in cellular structure and function. This antibody can be used in techniques such as Western blotting, immunocytochemistry, and ELISA to analyze beta-actin levels in various cell and tissue samples.
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Lab products found in correlation
Laemmli sample buffer, by Bio-Rad (1 mentions)
Cell lysis buffer, by Cell Signaling Technology (1 mentions)
Succinate dehydrogenase sdha, by Cell Signaling Technology (1 mentions)
Hrp conjugated anti rabbit secondary antibody, by Cell Signaling Technology (1 mentions)
Anti acetylated lysine, by Cell Signaling Technology (1 mentions)
2 protocols using anti human beta actin
1
Western Blot Analysis of PBMCs
2
Immunoprecipitation and Western Blot Analysis
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Total protein extraction and western blot analysis were performed as previously described [9] . Steps of immunoprecipitation assays were as follows: treated cells were resuspended in IP lysis buffer on ice for 5min, and then centrifuged at 14,000 g for 15 min. The supernatants were precleared with protein A/Gcoupled agarose (United States Santa cruz, #sc-2003), and subsequently incubated with 2 μg of the indicated antibodies and 20μl protein A/G agarose overnight at 4℃. After washing 5 times with lysis buffer, immunoprecipitates were boiled in 5× loading buffer for western blot analysis as mentioned. Western blot used the following primary antibodies: anti-human SIRT3, anti-acetylated-lysine, anti-human E-cad and anti-rabbit conformational speci c IgG from Cell Signaling Technology, anti-human N-cad from BD Company, anti-human beta-actin form Sigma.
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