Antibody against pml

Sedimentation experiments were conducted using a Beckman XL-I Optima analytical ultracentrifuge equipped with absorbance optics. Sedimentation studies were carried out at 200,000g, at 25 °C overnight. Three-channel with quartz windows were filled with 400 μl of sample (20 mM Tris, 100 mM NaCl, pH 8.0, with/without 1 mM DTT) at the concentration of 1 mg ml⁻¹. To investigate how protein concentration might influence PML RING tetramerization, the wild type protein at the higher concentration (5 mg ml⁻¹) was also tested. Absorbance profiles were acquired at a wavelength of 335 nm, chosen according to the protein concentration. Data analysis was carried out using SEDFIT^{40 (link)}, which employs the continuous c(s) conformational change model based on the Lamm equation, to determine the sedimentation coefficient distribution.
In order to check RING tetramerization in the context of full-length PML protein, gel filtration analysis was used. Recombinant proteins including PML∆BC (i.e., the deletion of residues, 120–360) and mutants were obtained by in vitro translation (Promega) using pcDNA3.1(−) vector with T7 promoter according to manufacturer’s standard protocol. The sample was then subjected to gel filtration analysis using Superose 6 and Superose 12 columns (GE) at a flow rate of 0.4 ml min⁻¹. Each fraction was monitored by Western blot using antibody against PML (Abcam).