Anti-IRS1 pS307 Antibody - Western Blot. Western blot of Affinity Purified anti-IRS1 pS307 antibody shows detection of a band at ~180 kD believed to represent phosphorylated IRS1 (arrowhead). Lane 1 shows staining of human 293 cell lysate. Lane 2 shows staining of 293 cell lysate prepared from cells serum-starved for 18 h followed by treatment with 5 ug/ml of anisomysin for 30 min. The pronounced staining of the band at 180 kD is not seen when the antibody was pre-incubated with immunizing peptide prior to reaction (data not shown). The identity of the intensely reactive bands at ~70 kD in both lane 1 and 2 is unknown, although these bands were also competed out by pre-incubation with the immunizing peptide. Approximately 25 ug of each lysate was separated on a 4-20% Tris-Glycine gel by SDS-PAGE and transferred onto nitrocellulose. After blocking with 5% goat serum, 0.5% BLOTTO in PBS, the membrane was probed with the primary antibody diluted to 1:250. Reaction occurred overnight at 4C followed by washes and reaction with a 1:10000 dilution of IRDye800 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). IRDye800 fluorescence image was captured using the Odyssey Infrared Imaging System developed by LI-COR. IRDye is a trademark of LI-COR, Inc. Other detection systems will yield similar results.