NanoPhotometer
®
NP80/N60/N50/C40
User Guide Version 3.1
53
P
ROTEIN
UV
M
ETHOD
O
VERVIEW
The Protein UV method exploits the inherent absorbance of proteins at 280 nm in combination
with the Beer-Lambert Law, where each protein is characterized by a protein specific extinction
coefficient (
ε) which can be used to determine total protein concentration of a solution. The
intrinsic absorbance of proteins is due to the presence of aromatic amino acids in their structure,
primarily tryptophan and tyrosine, as well as cysteine (oxidized cysteine residues in a disulphide
bond). The aromatic amino acid residues in a protein containing tryptophan and tyrosine exhibit
strong intrinsic absorbance at 280 nm, with a lesser contribution by phenylalanine. Therefore, it
is the aromatic amino acid residues which dictate the extinction coefficient at 280 nm for a
protein.
The most straightforward method to determine concentration of a purified, homogenous protein
with a known extinction coefficient (
ε) is by direct measurement of UV280
provided as long as
the protein contains no prosthetic groups with strong absorption in the same region. However,
for unknown proteins including homogenous protein mixtures, it is possible to make direct A
280
measurements
using a composite ε value derived from comparison of many proteins, although
this will only provide an approximate but close estimate of the true protein concentration.
The NanoPhotometer
®
determines protein concentration by performing calculations based on
specific ε values, either pre-programmed in the instrument or entered manually by the user.
Extinction coefficient (
ε) values at 280 nm vary greatly for different proteins due to their
particular aromatic amino acid content. Fixed ε values are pre-programmed in the software for
certain proteins (see Table 3). However, if the protein of interest is not included in the pre-
programed methods it is also possible to manually enter the specific
ε for the protein of interest
using the Mol. Ext. Coefficient or Ext. Coefficient protein option. For correct calculation, it is
necessary to supply either: a) the
mass extinction coefficient (ε in l/g*cm); or b) the molar
extinction
coefficient (ε
M
in M
-1
*cm
-1
), and the molecular weight of the protein expressed in
molar mass units (g/mol).
To determine the degree of dye labelling of a protein, the absorbance measured at the
wavelength corresponding to the absorbance maximum of the fluorescence dye is used (see
Table 3). The corresponding extinction coefficient of the dye is used along with the Beer-
Lambert Law to determine the dye concentration.
Note:
It is important to ensure the extinction coefficient and units entered are correct in order to
ensure that calculations are performed properly for accurate concentration values.
Sample Control™ gives useful information about air bubbles, sample impurities, turbidity, lint
residues and potential contaminations. If the Sample Control™ detects any inconstancy an alert
icon
is shown in the result/table area. A click on the alert icon shows additional information
about the inconstancy.
