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20
English
Issue 02 - 07/2001
UV methods
Protein Impurity
The presence of nucleic acid in a protein solution can have a significant effect due to
strong nucleotide absorbance at 280 nm. To compensate for this by measuring Abs
260, the equation of Christian and Warburg for the protein crystalline yeast enolase
(Biochemische Zeitung 310, 384 (1941)) can be applied:
Protein (mg/ml) = 1.55 * Abs 280 – 0.76 * Abs 260
This equation can be applied to other proteins if the corresponding factors are
known. To customise the equation for a particular protein, the absorbances at 260
and 280 nm should be determined at known protein concentrations to generate simple
simultaneous equations; solving these provides the two coefficients. In cases where
Factor 2 is found to be negative, it should be set to zero since it means there is no
contribution to the protein concentration due to absorbance at 260 nm. The use of
background correction at 320 nm is optional.
Protein (mg/ml). = (Factor 1 * Abs 280) – (Factor 2 * Abs 260)
Set Factor 2 = 0.00 for direct
λ
280 UV protein measurement; Factor 1 is based on the
extinction coefficient of the protein. If BSA (bovine serum albumin) is an acceptable
standard, setting Factor 1 = 1.115 will give linear results from 0 to 0.8 mg/ml protein.
Protein (mg/ml) = 1.115 * Abs 280
Rapid measurements such as this at Abs 280 are particularly useful after isolation of
proteins and peptides from mixtures using spin and HiTrap columns by centrifuge
and gravity, respectively.
The procedure is as follows:
•
Select if background correction at 320 nm is required using
4
•
Enter Factor 1
•
Enter Factor 2
•
Save method if required using
4
•
Insert reference and samples, and press
run
