QUANTITATIVE DENSITOMETRY OF PROTEINS STAINED WITH COOMASSIE-BLUE USING A HEWLETT-PACKARD SCANJET SCANNER AND SCANPLOT SOFTWARE

In the present study we evaluated the performance of a software/scanne r system that employed the Hewlett Packard (HP) ScanJet Plus and Scanp lot Software for densitometric quantification of protein loads stained with Coomassie Brilliant Blue following sodium dodecyl sulfate-polyac rylamide gel electrophoresis (SDS-PAGE). Gels with bovine serum albumi n (BSA) standards, ranging from 0.125 to 10 mu g, were scanned using r eflectance densitometry with 127 mu m step size in both the x and y di rections and a resolution of 200 dots per inch. Densitometric volume w as calculated for each protein band from scanner output in the tagged image file format (TIFF) by a customized software package, Scanplot V. 4.05 (Cunningham Engineering). Protein loads between 0.125 and 10.0 m u g vs. volume were fit by a second-order regression: Volume = -0.58 X protein load(2) + 16.82 X protein load + 7.87 (r = 0.991, p < 0.01). The same gels were scanned and quantified using a transmittance laser densitometer; densitometric volumes measured by both systems were high ly correlated (r(2) = 0.981, p < 0.01). Additional gels of BSA, smooth muscle myosin heavy chain (myosin), and actin displayed linear relati onships between protein loads up to 4.0 mu g and densitometric volume reflecting unique dye binding properties. We conclude that accurate an d reproducible quantitative densitometry of SDS-PAGE can be performed using the HP ScanJet Plus scanner and Scanplot software.