Protocol for the determination of protein using UV absorption.
Protein Determination by UV Absorption Protocol, ( Modified from the protocol by Alastair Aitken and Michèle P. Learmonth)
Materials for UV Protein Concentration
1. 0.1 M K2SO4 (pH 7.0).
2. 5 mM potassium phosphate buffer, pH 7.0.
3. Nonionic detergent (0.01% Brij 35)
5. 0.2-µm Millipore (Watford, UK) filter.
6. UV-visible spectrometer: The hydrogen lamp should be selected for maximum intensity
at the particular wavelength.
7. Cuvets, quartz, for <215 nm.
Protein Concentration UV Method
Estimation of Protein by Near UV Absorbance (280 nm):
1. A reliable spectrophotometer is necessary. The protein solution must be diluted in the
buffer to a concentration that is well within the accurate range of the instrument (see Notes 1 and 2).
2. The protein solution to be measured can be in a wide range of buffers, so it is usually no problem to find one that is appropriate for the protein which may already be in a particular buffer required for a purification step or assay for enzyme activity, for example (see Notes 3 and 4).
3. Measure the absorbance of the protein solution at 280 nm, using quartz cuvets or cuvets that are known to be transparent to this wavelength, filled with a volume of solution sufficient to cover the aperture through which the light beam passes.
4. The value obtained will depend on the path length of the cuvet. If not 1 cm, it must be
adjusted by the appropriate factor. The Beer-Lambert law states that: A (absorbance) = ε c l where ε = extinction coefficient, c = concentration in mol/L and l = optical path length in cm. Therefore, if ε is known, measurement of A gives the concentration directly, ε is
normally quoted for a 1-cm path length.
5. The actual value of UV absorbance for a given protein must be determined by some absolute method, e.g., calculated from the amino acid composition, which can be determined by amino acid analysis. The UV absorbance for a protein is then calculated according to the following formula: A280 (1 mg/mL) = (5690nw + 1280ny + 120nc)/M
where nw, ny, and nc are the numbers of Trp, Tyr, and Cys residues in the polypeptide of
mass M and 5690, 1280 and 120 are the respective extinction coefficients for these residues (see Note 5).
See also Protein Concentration Protocols from other labs.
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