EGFR Antibody (E-8): sc-374607

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	-Mouse (1:1000) -Use clean forceps to gently handle the blot from the corner without creasing the membrane. Do not write on the blot with pen or marker, as the ink can fluoresce and cause background.

Protocol tips
-Mouse (1:1000) -Use clean forceps to gently handle the blot from the corner without creasing the membrane. Do not write on the blot with pen or marker, as the ink can fluoresce and cause background.

Publication protocol

Cells were harvested at 80% confluence into radio-immunoprecipitation assay (RIPA) buffer containing protease inhibitors (Roche Diagnostics, Germany). The cellular debris was pelleted by centrifugation (20,000 ×g for 5 min) and protein concentration quantified using the Lowry protein assay. Equal quantities of protein (20 μg) heat-denatured in the presence of 0.03% v/v β-mercaptoethanol and 1X NuPAGE LDS Sample Buffer (Fisher Scientific, Loughborough, UK) in a total of 30 μl was loaded onto a NuPAGE Novex 4–12% gradient Bis-Tris gel (Fisher Scientific, Loughborough, UK) and separated by electrophoresis. Western blotting was performed by transfer of the separated proteins by electroblot to a (0.22 μm) PVDF (polyvinylidene fluoride) membrane (BioRad, Hemel Hempstead, UK), the membrane was blocked using 2.5% w/v BSA in PBS − 0.05% v/v Tween-20 for 30 min.
Protein expression levels were confirmed using primary anti-HER2/Neu mouse monoclonal antibody (Santa Cruz; Dallas, Texas, US sc-08, 1:500 dilution in 1% w/v BSA-Tween) and anti-EGFR mouse monoclonal antibody (Santa Cruz; Dallas, Texas, US, sc-374607 1:1000 dilution in 1% w/v BSA-Tween) followed by goat anti-mouse horseradish peroxidase conjugated antibody (Santa Cruz Dallas, Texas, US sc-2005; 1:2500 dilution in 1% v/v BSA-Tween) at room temperature. The HRP reaction was detected using ClarityTM Western ECL substrate and visualised using ChemiDoc XRS with Image Lab software (Bio-Rad, Hemel Hempstead, UK).

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