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In biochemistry, the molar absorption coefficient of a protein at 280 nm depends almost exclusively on the number of aromatic residues, particularly tryptophan, and can be predicted from the sequence of amino acids. [6] Similarly, the molar absorption coefficient of nucleic acids at 260 nm can be predicted given the nucleotide sequence.
The Warburg–Christian method is an ultraviolet spectroscopic protein and nucleic acid assay method based on the absorbance of UV light at 260 nm and 280 nm wavelengths. Proteins generally absorb light at 280 nanometers due to the presence of tryptophan and tyrosine. Nucleic acids absorb more at 260 nm, primarily due to purine and pyrimidine ...
The ratio of the absorbance at 260 and 280 nm (A 260/280) is used to assess the purity of nucleic acids. For pure DNA, A 260/280 is widely considered ~1.8 but has been argued to translate - due to numeric errors in the original Warburg paper - into a mix of 60% protein and 40% DNA. [ 6 ]
[1] [2] Most proteins absorb at 280 nm due to the presence of tyrosine and tryptophan. Of the aromatic amino acids, tryptophan has the highest extinction coefficient; its absorption maximum occurs at 280 nm. The absorption maximum of tyrosine occurs at 274 nm. [3]
Specialized fluorescent dyes bind specifically to the substances of interest. A spectrophotometer is used in this method to measure the natural absorbance of light at 260 nm (for DNA and RNA) or 280 nm (for proteins). [5] [6] [7] [8]
The anionic bound form of the dye which is held together by hydrophobic and ionic interactions, has an absorption spectrum maximum historically held to be at 595 nm. [5] The increase of absorbance at 595 nm is proportional to the amount of bound dye, and thus to the amount (concentration) of protein present in the sample. [6]
Absorbance: Read at 280 or 215 nm. Can be very inaccurate. Detection in the range of 100 μg/mL to 1 mg/mL. Ratio of absorbance readings taken at 260/280 can indicate purity/contamination of the sample (pure samples have a ratio <0.8) Bradford protein assay: Detection in the range of ~1 mg/mL; Biuret Test Derived Assays:
An absorption peak at 280 nm is visible due to aromatic amino acids which transfer their excitation energy to the green chromophore. The quantum yield of the green-emitting form of Eos is 0.7. [ 2 ] In the red shifted species, there are pronounced vibronic sidebands separate from the main peak at 533 nm and 629 nm in the excitation spectrum and ...