NanoPhotometer
®
N120/NP80/N60/N50/C40 User Manual Version 4.3.1
80
C =A
280
* ε
prot
* Ð
C Concentration in mg/ml
A
280
Absorbance at 280 nm (10 mm path)
A
BKG
Absorbance at selected background wavelength (10 mm path)
A
max,dye
Absorbance value at the absorbance maximum of the dye (10 mm path)
ε
prot
Extinction coefficient/protein factor in g*cm/l
cf
dye
Dye-dependent correction factor at 280 nm
Ð Dilution factor
Dye Concentration
For dye-labeled proteins, the concentration of the dye is calculated using a modified form of
the Beer-Lambert equation. For these calculations, the instrument considers the absorption
maximum of the dye, and a dye-specific extinction coefficient (see Table 4 on page 82). The
dye concentration is calculated with or without background correction for are as follows:
With background correction:
C =
(A
max,dye
A
BKG
) * Ð
ε
dye
* 10
6
Without background correction:
C =
A
max,dye
* Ð
ε
dye
* 10
6
C Concentration
A
max, dye
Absorbance at the max dye absorbance value (10 mm path)
A
BKG
Absorbance at selected background wavelength (10 mm path)
ε
dye
Extinction coefficient of dye in M
-1
*cm
-1
Ð Dilution factor
Degree of Labeling (DOL)
DOL is the degree of labeling based on the average number of dye molecules coupled to a
protein molecule. The degree of labeling can be determined from the absorption spectrum of
the labeled antibody with or without background/dye correction as follows:
With background and with dye correction:
DOL =
(A
max, dye
A
BKG
) * ε
prot
A
280
A
BKG
cf
dye
* (A
max, dye
A
BKG
) ε
dye
With background and w/o dye correction:
DOL =
(A
max, dye
A
BKG
* ε
prot
(A
280
A
BKG
) * ε
dye
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