The Protein Purification Facility
The Wolfson Centre for Applied Structural Biology 
The Hebrew University of Jerusalem
Dr. Mario Lebendiker
mariol@mail.ls.huji.ac.il  Tel: 972-2-6586920 

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


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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Dr. Mario Lebendiker The Protein Purification Facility
The Wolfson Centre for Applied Structural Biology,    The Hebrew University of Jerusalem
mariol@mail.ls.huji.ac.il  Tel: 972-2-6586920  

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