3. While the binding of Coomassie blue to proteins can lead to accurate measurements of protein concentration, there are situations where the absorbance values could be misleading. For example, detergents are often used in the purification of proteins. Certain detergents mixed in with the dye could lead to incorrect determinations of protein concentration. Take a look at the spectrum of the detergent polysorbate 80, above. If you had used this detergent to purify the protein in part c above, would you expect it to interfere with your concentration calculations? Why or why not?
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Proteins absorb strongly at 280 nm due to the presence of aromatic amino acids (Phenyl alanine, tyrosine and tryptophan). The peptide bonds found in the amino acids also can absorb at 205 nm. The quantitation of the proteins by absorbance assay is usually performed by measuring the absorbance at 280 nm.
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