(37d) Quantification of 2D Gel Western Blot Overlay Images

Previously, we have shown that epidermal growth factor receptor (EGFR) may be detected in human lung cancer samples with an anti-EGFR antibody using 2D gel western blotting. Furthermore, EGFR activated by tyrosine phosphorylation may be distinguished from inactive by colorizing and overlaying EGFR/pTyr western blot images from the same 2D gel. Visual comparison of overlay images shows the active EGFR is dramatically higher in some but not all tumors compared to the corresponding control lung tissue. But visual comparisons do not suffice when many samples are tested. In that case it is desirable to quantify images and express differences as a numerical value. The highly sensitive ECL/chemiluminescence used for EGFR is known to vary dramatically with time and amount of horseradish peroxidase (HRP) which oxidizes luminol to produce light. In this report we describe creation of an internal standard for chemiluminescence detection. The Lightning-Link HRP conjugation kit from Innova Biosciences was used to attach HRP to a purified protein, tropomyosin. In this case, no primary antibody is needed for western blotting, the HRP-tropomyosin reacts in the system to produce light exactly as the HRP-secondary antibody does in a normal western blot. Linearity of the HRP-tropomyosin (HRP-T) chemiluminescent signal within a 1D gel was demonstrated by loading varying amounts of HRP-T in different lanes and plotting band density versus concentration. Results are presented for quantification of EGFR protein spots detected by 2D western blotting in tumor and control samples using Progenesis SameSpots software with and without normalization to the internal HRP-T standard.