Gel doc xr plus system

Sample lysates of the hippocampus were blended with 4× Laemmli sample buffer (Bio-Rad laboratories, Hercules, CA, USA) and 10% β-mercaptoethanol (Merck, Darmstadt, Germany) and diluted in PBS to equalize their protein concentration and heated (95 °C) for 5 min. Total protein (20 µg/well) was loaded on 4–20% Criterion TGX Stain-Free Precast Gels (Bio-Rad laboratories, Hercules, CA, USA) and transferred onto 0.2 µm nitrocellulose membranes (Bio-Rad laboratories, Hercules, CA, USA). Membranes were blocked in TBS-T (pH 7.45, Tris-Base, NaCl, 0.1% Tween 20) containing 5% nonfat milk for 60 min and then incubated in the following primary antibodies overnight at 4°C: SIRT1 mouse monoclonal antibody (1:500 dilution, CST 8469, Cell Signaling, Leiden, WZ, Netherlands) and SIRT6 rabbit monoclonal antibody (1:500 dilution, CST 12486, Cell Signaling, Leiden, WZ, Netherlands). The following day, membranes were placed with secondary antibody (1:5,000, Vector Laboratory, Burlingame, CA, USA) in 5% nonfat milk for 1 h and the bands were visualized using a Clarity Western ECL substrate (Bio-Rad laboratories, Hercules, CA, USA) and a Gel Doc XR Plus system (Bio-Rad laboratories, Hercules, CA, USA). Immunoreactive bands were quantified using Image Lab Software (Bio-Rad laboratories, Hercules, CA, USA) and normalized to total protein.

The parasite somatic proteins were precipitated with TCA/Acetone (10% w/v) in 1:1 ratio over night at -20°C and then centrifuged at 10,000×g for 30 minutes at 4°C. The supernatant was discarded and the pellet was washed four times with acetone and protein content was estimated by the dye binding method [41 ]. The SDS PAGE was carried out according to the method of Laemmli (1970) [45 (link)], using a 12% separating and a 5% stacking gel. The electrophoresis was carried out at a constant voltage of 100V on a Bio-Rad Mini-Protean Tetra System. Thereafter, the gel was stained with 0.25% Coomassie Brilliant Blue-R250 (CBBR- 250) dye prepared in methanol, water, acetic acid (50:40:10) solution. The image was taken on Gel Doc XR Plus system (Bio-Rad, U.S.A.).

Cells were seeded in a 6-well plate at a density of 1.5 × 10⁶ per mL. A total of 4.5 × 10⁶ cells were seeded per well and treated with different bortezomib concentrations for 24 h. After treatment, the cells were harvested, and genomic DNA was extracted according to the package insert of DNeasy Blood and Tissue Kit QIAGEN (Cat. No. 69504). The recovered DNA concentration was measured using Nanodrop. The DNA fragmentation was analyzed by 1% Agarose Gel electrophoresis using a Bio-Rad system (Voltage 180, Time 1:15 h). Gel was visualized using Gel-Doc XR plus system (Bio-Rad) and documented.