| THEORY & FORMULAE |
With the aid of spectroscopy, the quantitative analysis of nucleic acids and proteins is now an established method in many laboratories.
Proteins absorb Ultraviolet light mainly of 280 nm wavelength. The reading at 280nm allows calculation of the concentration of nucleic acid in the sample and in the original stock. The absorbance A280 (also referred to as: optical density, OD) of 1 corresponds to approximately 1 mg/ml for uncontaminted proteins.
Proteins also absorb light at at 205 nm and with a greater sensitivity than at 280nm. Quantification using 280 nm works best for protein in the range 0.02 to 3mg in a 1-ml cuvette, and 205m works best in the 0.001 - 0.1 mg/ml range. Although nucleic acids (e.g. DNA and RNA) maximally absort UV light at 260 nm, they do show strong absorbance at 280 nm and to a lesser degree at 205 nm. Thus in the presence of nucleic acids, it is necessary to correct the protein measurements at 280 and at 205 nm.
Some proposed equations are implemented here:
A280 Readings ignoring contamination: Stock Concentration = A280 x [Dilution factor]
A280 Readings with contamination (Layne, 1957): Stock Concentration = [1.55A280 - 0.76260] x [Dilution factor]
A205 Readings ignoring contamination (Stoscheck, 1990): Stock Concentration = [1/31] x A205 x [Dilution factor]
A205 Readings with contamination (Peterson, 1983): Stock Concentration = A205/[27 + 120A280/A205] x [Dilution factor]
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