A rapid solid-phase fluorescence-based protein assay for quantitation of protein electrophoresis samples containing detergents, chaotropes, dyes, and reducing agents

Abstract

A new solid-phase, fluorescence-based protein assay was developed that quantifies proteins in the presence of detergents, urea and reducing agents (one-dimensional sodium dodecyl sulfate (1-D SDS) lysis buffers and urea-isoelectric focusing (IEF) buffers). A specially designed 96-well microplate facilitates application of protein samples to the assay paper and allows easy quantitation of samples using fluorescence microplate readers (top or bottom reading format). Alternatively, stained membranes may be directly scanned using a variety of different laser or charge-coupled device (CCD)-based imaging devices with UV or visible imaging capabilities. Since protein is specifically bound to the membrane, contaminants are readily washed away, avoiding interference with the protein measurement. The protein assay has a dynamic range extending from 10 ng to 5 μg of protein per microliter and requires only 1 μL of sample, which is ideal for samples destined for electrophoresis. The protein-to-protein variability of staining of ten different proteins was determined to be comparable with that of the bicinchoninic acid (BCA) and Lowry assays (16%). Additionally, the quality of the assay according to Z-factor analyses is excellent. © 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.