Anti EGFR  

Immunohistochemistry of Anti-EGFR Antibody with positive staining.

Western blot using Rockland's anti-EGFR antibody. Lane 1: unstimulated A431 whole cell lysates (p/n W09-000-361). Lane 2: EGF stimulated A431 whole cell lysates (p/n W09-000-362). Shows detection of a band at ~170 kDa corresponding to human EGFR present in unstimulated and stimulated (50 ng/ml for 15 min) lysates (arrowhead). Loaded: 30μg lysate was resolved on a 4-20% Tris-Glycine gel by SDS-PAGE and transferred onto nitrocellulose. Primary Antibody: Anti-EGFR at 1:1,000 overnight at 4° C. Secondary Antibody: IRDyeR 800 conjugated Gt-a-Rabbit IgG (H&L) MX10 (p/n 611-132-122) at 1:10,000 dilution of 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). IRDyeR 800 fluorescence image was captured using the OdysseyR Infrared Imaging System developed by LI-COR. IRDye is a trademark of LI-COR, Inc. Other detection systems will yield similar results.

Combined immunoprecipitation and western blot using anti-EGFR antibody. Lysates were prepared from GN4 rat liver epithelial cells both with (+) EGF treatment for 15' at 100 ng/ml and without (-) the addition of EGF. The combination of immunoprecipitation and western blotting was performed using the anti-EGFR antibody for immunoprecipitation (10 μL) followed by western blot detection using an anti-phosphotyrosine antibody (Panel A). This was repeated in reverse order using a 1:2000 dilution of anti-EGFR for western blot (Panel B). Visualization occurred using an ECL system. Film exposure was approximately 1’. Other detection systems will yield similar results.