Biotinylated anti egfr antibody

Epidermal growth factor receptor (EGFR) was measured with a sandwich ELISA utilising goat polyclonal antibodies against the extracellular domain of EGFR (R&D Systems Europe Ltd, Abingdon, UK, Cat no. DYC1854-5). All urine samples were measured using 50 μl of urine plus 50 μl of 1% BSA in PBST per well. Urinary EGFR is expressed as pg EGFR per mg creatinine (determined by the Jaffe method). Cell lysates were prepared in PBS containing 1% Igepal CA-630 and Complete Protease Inhibitor Cocktail (Roche Diagnostics Ltd, Burgess Hill, UK) and the protein concentrations determined using the bicinchoninic acid assay calibrated with bovine serum albumin. Secretome and lysate EGFR concentrations were determined using appropriate volumes of sample (2–100 μl) for the concentration range of the assay. Secretome and lysate EGFR concentrations are expressed as ng EGFR per mg of total cell protein. For western blotting, proteins were separated with SDS–PAGE on 4–12% gels using MOPS running buffer (Life Technologies, Paisley, UK), blotted onto PVDF and probed with 0.2 μg ml⁻¹ biotinylated anti-EGFR antibody (R&D Systems Europe Ltd, Cat no. BAF231) followed by streptavidin-HRP.