Anti-Rad9 Antibody- Western Blot. Affinity purified phospho-specific antibody to yeast Rad9 at pS1260 was used at a 1:200 dilution incubated overnight at 4° C to detect Rad9 by Western blot. Lanes were loaded with 50 ng each of recombinant GST fusion protein containing S. cerevisiae Rad9 (aa ~60 kD) 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. Rad53 kinase autophosphorylates and appears cross reactive as it is detected on the blot as 90 and 110 kD bands (asterisk). Detection occurred using a 1:5000 dilution of IRDye800 conjugated Donkey anti-Rabbit IgG (code # for 1h at room temperature. LICORs Odyssey Infrared Imaging System was used to scan and process the image. Other detection systems will yield similar results.