Western blot using Affinity Purified anti-Yeast CHK1 antibody shows detection of a bands corresponding to CHK1 in Saccharomyces cerevisiae lysates. Two strains of S.cerevisiae were tested. Lane 1 shows a predominant band at ~60 kDa. Lane 2 shows a predominant band at ~38 kDa. Specific band staining is blocked when antibody is preincubated for 45 min at room temperature with 50ug of peptide immunogen (lanes 3 and 4 respectively). Lysates were separated by 4-20% SDS-PAGE and transferred onto nitrocellulose. After blocking, the membrane was probed for 2 h at room temperature with the primary antibody diluted to 1:750 in blocking buffer diluted 1:5 in PBS. The membrane was washed and reacted with a 1:10,000 dilution of IRDye (TM)800 conjugated Gt-a-Rabbit IgG [H&L] MX for 45 min at room temperature (800 nm channel, green). Molecular weight estimation was made by comparison to prestained MW markers in lane M (700 nm channel, red). IRDye (TM)800 fluorescence image was captured using the Odyssey (R) Infrared Imaging System developed by LI-COR. IRDye is a trademark of LI-COR, Inc. Other detection systems will yield similar results.
