A multipurpose solid-phase method for protein determination with Coomassie brilliant blue G-250

Abstract

The multipurpose method for protein determination (MMPD) is based in the Coomassie brilliant blue G-250 binding to immobilized and washed proteins in paper filter disks, and the A 600 measurement of the eluted protein-dye complexes. The analysis requires 5-μl samples, put in 7-mm paper filter disks, which can be stored for up to 2 weeks, without sensible changes in their protein content. The A 600 is a logarithmic function of the log of bovine serum albumin quantity, between 2.5 and 250 μg with two linear segments used as standard curves; one between 2.5 to 20 μg and the other between 20 and 80 μg. Results with the MMPD were quantitatively comparable with those obtained with the method of Lowry et al., and those reported by other workers, assaying the following material: (i) bovine serum albumin, in doubly distilled water or in presence of several substances that interfere with the current methods; and (ii) total homogenates and their respective trichloroacetic acid-insoluble extracts, which were obtained from several phenol-rich vegetal specimens and complex animal products. The MMPD is proposed as an alternative for protein determination for its versatility and reliability, in both vegetal or animal products, especially when the analysis with traditional methods is made difficult by the presence of natural or added interfering substances and the sample volume is too small to discard them, or when the material must be stored for relatively long periods of time, prior to its processing.