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
1) Centrifuge non clear protein solutons for
5 minutes, 14000 rpm, prior to taking any readings
2) 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. Do not allow
any bubbles to stay in the light path. It is best to measure absorbances in the range 0.05-1.0
(use solvent as blank).
3) 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.
4) Protein concentration can be measured according to Beer-Lambert law:
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. The value obtained will depend
on the path length of the cuvet. If not 1 cm, it must be adjusted by the
appropriate factor.
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.)
5) UV Scan 340
– 220 nm.: A clean protein shows a symmetric curve with a maximum at 280
nm; while a shoulder at 320 nm could indicate aggregation.
Interfering Reagents for UV Absorbance 280nm
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Data were obtained from Stoscheck C.M. 1990 Quantitation
of protein. Methods in Enzymology 182: 50-68.
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Copyright
©, 2002, The Hebrew University of Jerusalem. All Rights Reserved.