Denaturing PAGE, in-gel fluorescence, immunoblotting and autoradiography

Sample preparation including cold acetone precipitation followed by denaturing PAGE using NuPAGE 10% Bis-Tris and 3–8% Tris-Acetate precast gels (Life Technologies) as well as detection of in-gel fluorescence and autoradiography were performed as described previously and in accordance to the manufacturer’s instructions⁶. Due to increased protein concentrations resulting from utilization of the CrPV-IRES the amount of sample applied to the denaturing page was reduced to correspond to 2.5 μl of the initial fractions from the cell-free reactions.

Immunoblotting was performed using the “IBlot Gel Transfer Device” (Life Technologies) according to the manufacturer’s instructions. Following denaturing PAGE (3–8% Tris-Acetate) proteins were transferred to a PVDF membrane (Life Technologies). The membrane was blocked in “Roti-Block” (Roth) for 4 hours and subsequently incubated with “EGF Receptor (D38B1) XP^® Rabbit mAb 4267” or “Phospho-EGF Receptor (Tyr1068) (D7A5) XP^® Rabbit mAb 3777” primary antibodies diluted 1:1000 over night at 4 °C. “Anti-rabbit IgG, HRP-linked Antibody 7074” diluted 1:2000 was used as a secondary antibody and detection was carried out using the “Amersham ECL Prime Western Blotting Detection Reagent” (GE Healthcare) and the “Typhoon Trio + Variable Mode Imager” (GE Healthcare). All antibodies were purchased from “Cell Signaling Technology Inc.”. After detection, blotting membranes were dried and subjected to autoradiography.