This application note provides details about the quantification of protein content by measuring its absorbance at 280 nm using a METTLER TOLEDO UV/VIS spectrophotometer.
Background
Protein quantification is the determination of the total protein content in a sample or formulated product. True, decisive protein quantification is important because a range of other critical assays require precise total protein content results.
Proteins in solution offer a characteristic ultraviolet absorption at 280 nm due to the presence of the amino acids tyrosine and tryptophan. Quantification of protein by directly measuring its absorbance at 280 nm is a fast and convenient method for quantification since no additional reagents and/or incubations are required.
Details about the Protein
Different proteins have widely varying extinction coefficients depending on amino acid composition. To obtain high accuracy when determining protein concentration, the exact amino acid sequence of the analyzed protein must be known and the protein-specific absorption coefficient calculated before determining the concentration of the solution.
Parameters such as pH, ionic strength, etc. can change the absorbance spectrum and therefore, the protein sample is dissolved in Tris-EDTA buffer for improved stability. If a protein does not contain any tyrosine, tryptophan, and phenylalanine residues, it will have no absorbance at 280 nm and can therefore not be measured with the direct method at 280 nm.
Download our free application note to learn more and benefit from our experience determining protein concentration by UV Vis spectroscopy.
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