Bio spin column

Highly purified p53 was incubated with a ³²p-labelled probe (190 base pairs) containing p53-binding element of ELOVL3 promoter or mutant ELOVL3 promoter in 1× binding buffer (10 mM Hepes, pH 7.6, 40 mM NaCl, 50 µM EDTA, 6.25% Glycerol, 1 mM MgCl₂, 1 mM Spermidine, 1 mM DTT, 50 ng/µl BSA, 5 ng/µl sheared single strand salmon DNA) for 20 minutes at room temperature (RT). The complex was analyzed by 4% TBE-PAGE and visualized by autoradiography. The probe was obtained by PCR, labelled by T4 kinase (NEB, M0201S) and purified by Bio-Spin column (Bio-Rad, 732-6223).

To prepare proteins for mass spectrometry analysis, frozen liver tissues of three Sirt1^+/+ control mice and Sirt1^-/- mice were homogenized in PLC lysis buffer (50 mM HEPES [pH = 7.5], 150 mM NaCl, 10% glycerol, 1 mM EGTA, 1% triton x-100) containing trichostatin A and protease inhibitor cocktail (Roche). Six milligrams of liver extracts were resuspended in 50 mM NH₄HCO₃ [pH 8.5] using buffer exchange column (Bio-spin column, Biorad) and trypsinized with sequencing-grade trypsin (V5111, Promega) at an enzyme-to-substrate ratio of 1:50 for 16 hours at 37°C. Digested peptides were reduced with 5 mM DTT at 50°C for 30 min and alkylated with 15 mM iodoacetamide at room temperature for 30 min in darkness, and then quenched by 15 mM cysteine at room temperature for 30 min. Additional trypsin was added at an enzyme-to-substrate ratio of 1:100 for 4 hours at 37°C to ensure complete digestion. Trypsinized peptides were dried in a speedvac for 30 min at 30°C. To prepare acK-peptide standard, acetylated bovine serum albumin (BSA) (ICP6090, Immunechem) was trypsinized using the same method.

2-(4-Isothiocyanatobenzyl)-diethylenetriaminepentaacetic acid (SCN-Bn-DTPA; Macrocyclics, Dallas, TX, USA) was used for labeling NuB2 with ⁹⁰Y. SCN-Bn-DTPA in dimethylformamide was added to NuB2 at 5 mg/ml in 50 mM borate-buffered saline (pH 8.5) at the molar ratio of 5:1. After incubation at 37 °C for 24 h, DTPA–NuB2 was purified using a Bio-Spin column (Bio-Rad Laboratories, Hercules, CA, USA). For radiolabeling, 25–50 μl of a solution of ⁹⁰YCl₃ (37 MBq, Nuclitec, Braunschweig, Germany) was incubated with 50–100 μl of 0.25 M acetate buffer (pH 5.5) for 5 min at room temperature, followed by incubation with 100 μg of DTPA–NuB2 for 1 h at 40 °C. The ⁹⁰Y-labeled antibody was purified using a Bio-Spin column or PD-10 column. The radiochemical purity of ⁹⁰Y-NuB2 was confirmed as >95% by Tec-Control Chromatography Strips (Biodex Medical Systems, Shirley, NY, USA) developed with saline. Iodine-125-labeled antibodies were prepared according to standard protocols for the chloramine-T method.
Briefly, 740 kBq/2 μl of Na¹²⁵I (PerkinElmer, Waltham, MA, USA) and 1 μg of chloramine-T in 1 μl of 0.3 M phosphate buffer were added to 40 μg of mAb in 100 μl of 0.3 M phosphate buffer. The ¹²⁵I-labeled mAb was purified using a Bio-Spin column.