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UV Absorbance (280 nm) – Protein Determination
Simple and quick method to accurately quantitate total protein in purified material or approximately quantitate total protein in crude lysates or partial purified material.
(Protein Determination by UV Absorption - Alastair Aitken and Michale Learmonth - Protein Protocols in CD Rom - Humana Press, 1998)
Introduction
Quantitation of the amount of protein in a solution is possible in a simple spectrometer. Absorption of radiation in the near UV by proteins depends on the Tyr and Trp content (and to a very small extent on the amount of Phe and disulfide bonds). Therefore the A 280 varies greatly between different proteins; for a 1 mg/mL solution, from 0 up to 4 for some tyrosine-rich wool proteins, although most values are in the range 0.5-1.5 (Kirschenbaum, D. M. (1975) Molar absorptivity and A1%/1 cm values for proteins at selected wavelengths of the ultraviolet and visible regions. Anal. Biochem. 68, 465-484).The advantages of this method are that it is simple, and the sample is recoverable. The method has some disadvantages, including interference from other chromophores, and the specific absorption value for a given protein must be determined. The extinction of nucleic acid in the 280-nm region may be as much as 10 times that of protein at their same wavelength, and hence, a few percent of nucleic acid can greatly influence the absorption.
METHOD
Measure the absorbance of the protein solution at 280 nm, using quartz cuvets or cuvettes 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. The protein solution must be diluted in the buffer to a concentration that is well within the accurate range of the instrument. It is best to measure absorbances in the range 0.05-1.0 (use solvent as blank).
The protein solution to be measured can be in a wide range of buffers. Bovine serum albumin is frequently used as a protein standard; 1 mg/mL has an A 280 of 0.66. At low concentrations, protein can be lost from solution by adsorption on the cuvette; the high ionic strength helps to prevent this. Inclusion of a nonionic detergent (0.01% Brij 35) in the buffer may also help to prevent these losses.
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) = Ê.c.l
where Ê = extinction coefficient, c = concentration in mol/L and l = optical path length in cm. Therefore, if Ê is known, measurement of A gives the concentration directly, e is normally quoted for a 1-cm path length.
A rapid way to calculate the extinction coefficient is using the ProtPARAM tool from ExPASy. (Note: you need to know the protein's amino acid sequence in order to calculate the extinction coefficient.)
Interfering Reagents for UV Absorbance 280nm
|
REAGENT |
CONCENTRATION |
Ammonium
Sulfate |
>50% |
|
Brij 35 |
1% |
|
DTT |
3mM |
|
EDTA |
30mM |
|
Glycerol |
>40% |
|
KCl |
100mM |
|
ßME |
10mM |
|
NaCl |
>1M |
|
NaOH |
>1M |
Phosphate
buffer |
>1M |
|
SDS |
0.1% |
|
Sucrose |
2M |
|
Tris Buffer |
0.5M |
|
TRITON X-100 |
0.02% |
|
Urea |
>1M |
Data were obtained from Stoscheck C.M. 1990 Quantitation of protein. Methods in Enzymology 182: 50-68.
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