The amount of protein in a sample can be quantified by several kinds of spectrop

Question

The amount of protein in a sample can be quantified by several kinds of spectrophotometric methods. One method is based on the amount of light directly absorbed by the protein (no added reagents), while other methods utilize a chromophore-containing reagent and measure light absorbed by protein-reagent complexes. Match the descriptions with the appropriate method.

Sapling Learning The amount of protein in a sample can be quantified by several kinds of spectrophotometric methods. One method is based on the amount of light directly absorbed by the protein (no added reagents), while other methods utilize a chromophore-containing reagent and measure light absorbed by protein-reagent complexes. Match the descriptions with the appropriate method Measures absorbance of protein-reagent complexes Measures protein absorbance Applies to both methods directly Detection wavelength of 280 nm, for aromatic side chains. Absorbance intensity is highly rp and Tyr residues detection wavelength so the maximum Absorbance intensity is independent Sample concentration is determined by comparison to a standard curve of protein amino acid composition generated with a pure protein. t on presence of Absorbance is proportional to solute concentratiorn Detection wavele set at Nucleic acids absorbight atmaximum absorbance for bound dye for a fixed path length. correction factor must be applied

Explanation / Answer

Measures protein absorbance directly

Absorbance intensity is highly dependent on presence of Trp and Tyr residues. (aromatic acids contribute for absorbance at 280 nm and independent to any protein-dye binding)

Nucleic acid absorbs light at detection wavelength so correction factor must be applied (as method is non-specific in nature; obtained absorbance is cumulative of all components present in solution, hence, correction factor is required)

Detection wavelength of 280 nm, the maximum absorbance for aromatic side chains (as no dye is present, absorbance is recorded at absorption wavelength of aromatic acids)

Measures absorbance of protein-reagent complexes

Detection wavelength set at maximum absorbance for bound dye (dye is only used in protein-reagent complexes)

Absorbance intensity is independent of protein amino acid composition (as dye is not specific to any amino acid, hence, amino acid composition is not crucial. In contrast, direct protein absorbance method relies on Trp and Tyr residues)

Standard concentration is determined by comparison to a standard curve generated with a pure protein (A standard curve is required to determine unknown protein concentration, eg. Lowry, Bradford method)

Applies to both methods

Absorbance is proportional to solute concentration for a fixed path length (This point relates to the principle of the instrument i.e. Beer’s lambert law. Hence, it is applicable to both methods)

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