Comparison of the Coomassie brilliant blue, bicinchoninic acid and Lowry quantitation assays, using non-glycosylated and glycosylated proteins.
The concentrations of several non-glycosylated and glycosylated recombinant and native proteins were determined by three widely used colorimetric methods: Coomassie brilliant blue, bicinchoninic acid and Lowry, and, for comparison, by amino acid composition analysis. The colorimetric methods gave results differing from the values derived from the amino acid analysis, in some cases by up to 60%. For the non-glycosylated recombinant proteins, the results were in relatively good agreement with each other and with the values determined on the basis of the amino acid analysis. The Coomassie blue method was strongly dependent on the hydrophobicity of the individual protein. The bicinchoninic acid method gave results closest to those of the amino acid analysis. For the glycosylated proteins, both recombinant and native, the Coomassie blue assay gave values lower, whereas the two other methods gave values higher than those determined on the basis of the amino acid analysis. The concentration of a recombinant interferon gamma receptor produced in two differently glycosylated forms was underestimated by the Coomassie blue assay and overestimated by the bicinchoninic acid and Lowry methods, while for the non-glycosylated form of the same protein, the three colorimetric methods delivered comparable values. The results suggest a potential interference of protein glycosylation with the colorimetric assays.
Fountoulakis
M
M
F. Hoffmann-La Roche Ltd., Pharmaceutical Research-New Technologies, Basel, Switzerland.
Juranville
J F
JF
Manneberg
M
M
D002241:Carbohydrates / Q000737:chemistry; D003124:Colorimetry / Q000379:methods*; D006031:Glycosylation; D007202:Indicators and Reagents; D007700:Kinetics; D011506:Proteins / Q000032:analysis* / Q000737:chemistry; D011804:Quinolines; D011994:Recombinant Proteins / Q000032:analysis / Q000737:chemistry; D012394:Rosaniline Dyes
D002241:Carbohydrates; D007202:Indicators and Reagents; D011506:Proteins; D011804:Quinolines; D011994:Recombinant Proteins; D012394:Rosaniline Dyes; C047117:bicinchoninic acid; C004692:coomassie Brilliant Blue