Affinity purified antibody to yeast Rad9 (1pan reactive) was used at a 1:200 dilution incubated 8 h at room temperature to detect Rad9 by Western blot. Lanes were loaded with 50 ng each of recombinant GST fusion protein containing S. cerevisiae Rad9 (aa ~60 kDa) on a 4-20% Criterion gel for SDS-PAGE as follows: Lane 1 - non-phosphorylated wild type yeast Rad9, Lane 2 - in vitro phosphorylated wild type yeast Rad9, Lane 3 - non-phosphorylated S1129A/S1260A double mutant Rad9, Lane 4 - in vitro phosphorylated S1129A/S1260A double mutant. Phosphorylation of Rad9 was by treatment with ATP and Rad53 kinase. Detection occurred using a 1:5,000 dilution of IRDye®800 conjugated Donkey anti-Rabbit IgG for 1h at room temperature. LICOR's Odyssey Infrared Imaging System was used to scan and process the image. Other detection systems will yield similar results.