Western blots were performed as we described previously (Han et al., 2012 (link); Milner et al., 2015 (link); Vellimana et al., 2011 (link)). Briefly, brains were harvested 48 h after sham or SAH surgery. Tissue from the hemisphere ipsilateral to endovascular perforation was lysed in a buffer containing 10mM HEPES, 5mM MgCl2, 1mM DTT, 2mM EDTA, 2mM EGTA, 1mM PMSF, 1% Triton X-100, 0.5mM sodium orthovanadate, 0.1uM okadaic acid, 25mM beta-glycerophosphate and protease inhibitor cocktail (Sigma, St. Louis, MO). The following primary antibodies were used: Rabbit anti-SIRT1 (Millipore, Burlington, MA)(Gueguen et al., 2014 (link); Michan et al., 2014 (link)) and mouse anti-α-tubulin (Sigma, St. Louis, MO). α-tubulin was used as the loading control. Blots were subsequently incubated with anti-mouse or anti-rabbit horseradish peroxidase-conjugated IgG and visualized using an enhanced chemiluminescence kit (BioRad, Hercules, CA).
Rabbit anti sirt1
Manufactured by Merck Group
Sourced in United States
Rabbit anti-Sirt1 is a laboratory reagent used for the detection and quantification of the Sirt1 protein. It is a polyclonal antibody raised in rabbits against a specific epitope of the Sirt1 protein. This antibody can be used in various analytical techniques, such as Western blotting, immunohistochemistry, and immunoprecipitation, to study the expression and localization of Sirt1 in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor cocktail, by Merck Group (2 mentions)
Mouse anti β actin, by Merck Group (2 mentions)
Rabbit anti zo 1, by Thermo Fisher Scientific (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Enhanced chemiluminescence kit, by Bio-Rad (1 mentions)
Mouse anti α tubulin, by Merck Group (1 mentions)
Rabbit anti histone h3, by Abcam (1 mentions)
Mouse anti bax, by Santa Cruz Biotechnology (1 mentions)
Rabbit anti bcl 2, by Santa Cruz Biotechnology (1 mentions)
Rabbit anti occludin, by Proteintech (1 mentions)
Mouse anti p65, by Cell Signaling Technology (1 mentions)
Nuclear and cytoplasmic protein extraction kit, by Beyotime (1 mentions)
Ab206655, by Abcam (1 mentions)
Ecl chemiluminescence, by Bio-Rad (1 mentions)
Rabbit anti p16, by Proteintech (1 mentions)
Rabbit anti β actin, by Cell Signaling Technology (1 mentions)
Mouse anti β actin, by Cell Signaling Technology (1 mentions)
Ripa lysis buffer, by Merck Group (1 mentions)
Rabbit anti vinculin, by Cell Signaling Technology (1 mentions)
Rabbit anti β tubulin 9f3, by Cell Signaling Technology (1 mentions)
5 protocols using rabbit anti sirt1
1
Western Blot Analysis of SIRT1 in Subarachnoid Hemorrhage
2
Western Blot Analysis of Tight Junction and Apoptosis Proteins
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The protein levels of Occludin, ZO-1, SIRT1, Bax, Bcl-2, NF-κB p65, histone H3 and β-actin were detected by Western blotting. For p65, nuclear extracts were prepared using a Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime, Shanghai, China) according to the manufacturer’s protocol. The primary antibodies were as follows: rabbit anti-Occludin (1:2000, Proteintech Group, Chicago, IL, USA), rabbit anti-ZO-1 (1:500, Thermo Fisher Scientific, Waltham, MA, USA), rabbit anti-Sirt1 (1:3000, Millipore, Billerica, MA, USA), mouse anti-Bax (1:500, Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-Bcl-2 (1:1000, Santa Cruz Biotechnology), mouse anti-p65 (1:1000, Cell Signaling Technology, Boston, MA, USA), rabbit anti-histone H3 (1:1000, Abcam), and mouse anti-β-actin (1:5000, Sigma-Aldrich).
3
Immunoblotting Analysis of Cell Lysates
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Cell or tissue lysates were extracted for loading into 10% SDS-PAGE gels and immunoblotting was performed as previously described 34 (link). Primary antibodies, including rabbit anti-Cyp27b1/1α(OH)ase (Abcam, ab206655), rabbit anti-VDR (Abcam, ab3508), rabbit anti-Sirt1 (Millipore, 07-131), rabbit anti-p16 (Proteintech, 10883-1-AP) and rabbit anti-β-actin (Cell Signaling Technology, 8457S) were used for immunoblotting. Immunoreactive bands were visualized with ECL chemiluminescence (Bio-Rad) and analyzed by Image J.
4
Protein Extraction and Western Blot Analysis
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Front paw skin was pulverized in liquid nitrogen and sonicated in RIPA lysis buffer (Sigma-Aldrich) containing protease inhibitor cocktail (Sigma-Aldrich, P8340). The lysates were centrifuged at 12,000 g at 4 °C for 10 min. 10 or 20 μg supernatants were analyzed by Western blot. The band intensity was normalized to β-actin or β-tubulin. Primary antibodies used include rabbit anti-BDNF H-117 (Santa Cruz Biotechnology, sc-20981, 1:200), rabbit anti-BDNF 19-HCLC (ThermoFisher, #710306, 1:500), rabbit anti-SIRT1 (Millipore, 07-131, 1:1000), rabbit anti-acetyl FOXO1 Lys294 (ThermoFisher, PA5-154560, 1:500), mouse anti-FOXO1 3B6 (ThermoFisher, MA5-17078, 1:500), mouse anti-β-actin (Cell Signaling Technology, #3700, 1:1000), rabbit anti-β-tubulin 9F3 (Cell Signaling Technology, #2128, 1:1000), and rabbit anti-vinculin (Cell Signaling Technology, #4650, 1:1000).
5
Quantifying Protein Expression in Cells
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Cells were harvested and lysed in lysis buffer for protein extraction. Protein concentrations were measured by BCA protein assay kit (Thermo Scientific). Proteins were separated by SDS-polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes. The membranes were incubated with primary antibodies: rabbit anti-SIRT1 (Millipore), anti-BECN1 (Abclonal Technology), anti-LC3B (Cell Signaling Technology), anti-p16 (Abclonal Technology), anti-p21 (Abclonal Technology), anti-p53 (Abclonal Technology), and mouse anti-β-actin (Sigma) followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies (HRP-donkey-anti-rabbit and HRP-donkey-anti-mouse) (Santa Cruz Biotechnology). Signal was detected by an ECL kit. The experiments were performed three times.
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