Anti-iASPP Antibody - Western Blot. Western blot of affinity purified anti-iASPP antibody shows detection of a band at ~100 kD (arrowhead) corresponding to isoform 1 of iASPP in MCF7 whole cell lysates. Preincubation with immunizing peptide blocks specific band staining (data not shown). Approximately 35 ug of lysate was separated by 4-20% Tris Glycine SDS-PAGE. After blocking, the membrane was probed with the primary antibody diluted to 1:1500 in 5% BLOTTO/PBS overnight at 4C. The membrane was washed and reacted with a 1:10000 dilution of IRDye800 conjugated Gt-a-Rabbit IgG [H&L] ( for 45 min at room temperature (800 nm channel, green). Molecular weight estimation was made by comparison to prestained MW markers. 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.