Anti-EGFR pY1197 Antibody - Western Blot. Western blot using Affinity Purified anti-EGFR pY1197 antibody shows detection of a band at ~170 kD corresponding to human EGFR (arrowhead). Staining is not seen in unstimulated A431 cells (lane 1), but is seen when A431 cells are stimulated with EGF (50 ng/ml for 15 min) (lane 2). Approximately 30 ug of lysate was separated 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 1:250. Reaction occurred overnight at 4° C 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 Ddyssey Infrared Imaging System developed by LI-COR. IRDye is a trademark of LI-COR, Inc. Other detection systems will yield similar results.