Retinal tissue was homogenized in lysis buffer (ThermoFisher, Waltham, MA, USA) containing 1% phosphatase and protease inhibitor cocktail (Sigma-Aldrich). Protein concentration was measured using the Bio-Rad protein assay kit (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s recommendation. Proteins from whole rat retinal tissue and HuREC lysates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene difluoride (PVDF) membrane. The membrane was blocked using 5% skim milk and incubated with the following primary antibodies: anti-HDAC6 (Abcam, Cambridge, MA, USA), anti-Trx-1, anti-sirtuin 1 (SIRT1) and anti-albumin (all from Cell Signaling Technology). After incubation with horseradish peroxidase–conjugated secondary antibody (GE Healthcare, Pittsburg, PA, USA), bands were detected using the enzymatic chemiluminescence reagent, ECL (GE Healthcare). Subsequently, the membranes were stripped using stripping buffer (Bio-Rad) and re-probed with anti-β-actin antibody (Sigma-Aldrich) to assess equal loading. Scanned images of blots were used to quantify protein expression using NIH ImageJ software (http://rsb.info.nih.gov/ij/ ).
Anti sirtuin 1 sirt1
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-sirtuin 1 (SIRT1) is a lab equipment product that detects the presence and levels of the SIRT1 protein. SIRT1 is a nicotinamide adenine dinucleotide (NAD+)-dependent deacetylase enzyme that plays a role in various cellular processes, including metabolism, cell cycle regulation, and stress response.
Automatically generated - may contain errors
Lab products found in correlation
Protein assay kit, by Bio-Rad (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by GE Healthcare (1 mentions)
Lysis buffer, by Thermo Fisher Scientific (1 mentions)
Enhanced chemi luminescence (ecl), by GE Healthcare (1 mentions)
Stripping buffer, by Bio-Rad (1 mentions)
Anti β actin antibody, by Merck Group (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Anti trx1, by Cell Signaling Technology (1 mentions)
Anti hdac6, by Abcam (1 mentions)
Anti p53, by Cell Signaling Technology (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Ecl kit, by Beyotime (1 mentions)
Anti gapdh, by Cell Signaling Technology (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Anti hdac1, by Cell Signaling Technology (1 mentions)
2 protocols using anti sirtuin 1 sirt1
1
Retinal Protein Expression Analysis
2
Western Blot Analysis of Protein Expression
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Proteins were extacted using a Western Cell lysis Buffer kit (Beyotime Institute of Biotechnology) and the concentrations were determined by a bicinchninic acid (BCA) Protein Assay Kit (Beyotime Institute of Biotechnology). In total, 30 µg total proteins were loaded per lane. Proteins were separated via 10% SDS-PAGE and transferred to PVDF membranes (EMD Millipore). Following blocking with 5% non-fat milk at room temperature for 1 h, the membranes were incubated at 4˚C overnight with the following primary antibodies: Anti-androgen receptor (AR; cat. no. 5153; 1:1,000; Cell Signaling Technology, Inc.); Anti-p53 (cat. no. ab26; 1:1,000; Abcam), anti-acetylated p53 (Lys379; cat. no. 2570; 1:1,000; Cell Signaling Technology, Inc.), anti-HDAC1 (cat. no. 34589; 1:1,000; Cell Signaling Technology, Inc.), anti-sirtuin 1 (SIRT1; cat. no. 8469; 1:1,000; Cell Signaling Technology, Inc.) and anti-GAPDH (cat. no. ab8245; 1:1,000; Abcam). After washing by TBS with 0.05% Tween-20, the membranes were incubated with appropriate horseradish peroxidase-conjugated secondary antibodies (cat. no. ab7090 and ab97040; 1:5,000; Abcam) at room temperature for 1 h. Protein bands were visualized using an ECL kit (Beyotime Institute of Biotechnology).
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