How to determine the molar absorption coefficient of a protein experiment?
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No method of protein concentration determination is perfect because each method has different constrains. Interfering buffer components, contaminating proteins or reactivity of individual proteins and buffer components can influence the measurement and lead to false values. Therefore amino acid analysis is still considered to be the most accurate method for protein quantification, in particular when a highly purified protein sample is used. If accurate protein concentrations are critical, results from several methods or assays may need to be compared to trust the observed values.
Proteins and peptides containing aromatic amino acid side chains exhibit strong UV-light absorption, and each protein has a distinct UV spectrum. Proteins and peptides absorb UV-light proportional to their amino acid content and total concentration. Different proteins may have widely varying extinction coefficients. If a protein does not contain any tyrosine, tryptophan, and phenylalanine residues, it will not be detected.