Protein extract (20 μg) was separated by electrophoresis on denaturing polyacrylamide gels (SDS-PAGE) and transferred to a nitrocellulose membrane (BioRad). Nonspecific binding sites were blocked and the membrane was incubated overnight at 4 °C with the primary antibody (anti-α-tubulin 1:5000 Abcam; anti-Sirt3 1:1000 ThermoFisher Scientific; anti-SOD2 1:1000, Santa Cruz Biotechnology; anti-VDAC 1:1000 ThermoFisher Scientific; anti-Hsp70 1:200 Santa Cruz Biotechnology; anti-SIRT1 1:1000 Origen). The secondary antibody was conjugated to the enzyme horseradish peroxidase (HRP). For visualization of immunoreactive bands on the membrane a peroxide and luminol solution was applied (Millipore). After development of the photographic film, the reactive bands were quantified densitometrically using ImageJ software.
Anti sirt3
Manufactured by Thermo Fisher Scientific
Sourced in United States
Anti-Sirt3 is a laboratory reagent used for the detection and quantification of the Sirt3 protein. It is a highly specific antibody that can be used in various immunoassay techniques, such as Western blotting and immunoprecipitation, to study the expression and function of Sirt3 in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Luminol, by Merck Group (1 mentions)
Anti sod2, by Santa Cruz Biotechnology (1 mentions)
Anti α tubulin, by Abcam (1 mentions)
Anti hsp70, by Santa Cruz Biotechnology (1 mentions)
Anti β actin primary antibody, by Cell Signaling Technology (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Beyotime (1 mentions)
Chemidoc xrs system, by Bio-Rad (1 mentions)
Anti sirt5, by Proteintech (1 mentions)
2 protocols using anti sirt3
1
Western Blot Protein Analysis
2
Western Blot Analysis of Sirtuins
Check if the same lab product or an alternative is used in the 5 most similar protocols
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Cells and mice tissue were lysed in RIPA buffer with PMSF (Beyotime, Shanghai, China), and then protein was adjusted to the same level, added loading buffer and boiled at 100 °C for 5 min. A 50µg mass of protein lysates were loaded on 10% SDS-PAGE gels and transferred using PVDF membranes. After block, the membranes were incubated overnight with anti-SIRT3 (1:1000; Thermo, Waltham, MA, USA), anti-SIRT4 (1:1000; Thermo, Waltham, MA, USA), anti-SIRT5 (1:1000; Proteintech, Wuhan, Hubei, China) or anti-β-actin primary antibodies (1:1000; Cell Signaling Technology, Danvers, MA, USA), followed by incubation with appropriate secondary antibodies (1:10,000; Cell Signaling Technology, MA, USA) at 37 °C for 1h. The signal was visualized and captured with a ChemiDoc XRS+system (Bio-Rad, Hercules, CA, USA).
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