Western blot using the anti-IL-1RII antibody. This blot shows detection of an IL-1RII-GST fusion protein (~500 ng, lane 1, green, 800 nm channel). Minimal reactivity is observed against GST (data not shown). Protein was resolved on a 4-20% Tris-Glycine gel by SDS-PAGE and transferred onto nitrocellulose. After blocking, the membrane was probed with the primary antibody diluted to 1:1,000. Incubation was for 2 hrs at room temperature followed by washes and reaction with a 1:10,000 dilution of IRDye 800 conjugated Gt-a-Rabbit IgG (H&L) MX10 for 45 min at room temperature. Molecular weight markers are shown for size comparison (lane M, red, 700 nm channel). IRDye 800 fluorescence image was captured using the Odyssey Infrared Imaging System developed by LI-COR. Other detection systems will yield similar results.