Implen NanoPhotometer, User Manual, Page: 69 / 70

NanoPhotometer
®
P-Class User Manual
Version 2.1
Page 69 / 70
12.3 Protein quantification
For determination of protein concentration in solution the absorbance at wavelength 280 nm is used. The function
describing the concentration to absorbance relation is a modification of the Lambert-Beer equation.
c
prot.
= Abs. 280 * A280 factor * lid factor * dilution factor
With background correction:
c
prot.
= (Abs. 280 – Abs. 320) * A280 factor * lid factor * dilution factor
c
prot
protein concentration (mg/ml)
Abs. 280 absorbance (AU) of proteins
A280 factor Default setting is BSA; molecular weight
prot.
/ molar extinction coefficient (M
-1
*cm
-1
)
prot.
or
1 / extinction coefficient (l/g*cm)
lid factor virtual dilution factor 5, 10, 50, 100 and 250; dependent on the used dilution lid.
A280 factors pre-programmed:
BSA
Serum
albumin
(mouse)
Serum albumin
(human)
IgG Lysozyme
A280 factor 1.45 1.57 1.76 0.71 0.38
Molecular Weight [g/mol] 69,323.4 68,692.5 69,365.7 150,000 14,300
Molar extinction coefficient [M
-1
*cm
-1
] 47,790 43,780 39,310 210,000 37,500
12.4 Protein fluorescent dye incorporation
To determine the protein concentration and the dye concentration after labelling a modification of the Lambert-Beer
equation is used. Background correction is always calculated; possibility to switch the dye correction on and off.

Calculation of labelled protein concentration
c
prot
= (Abs. 280 – Abs. 320) * A280 factor * lid factor * dilution factor
With dye correction:
c
prot
= [Abs. 280 – Abs.320 - CF
dye
* (Abs.
max, dye
– Abs. 320)] * A280 factor * lid factor * dilution factor
c
prot
protein concentration (mg/ml)
Abs. 280 absorbance (AU) of proteins
CF
dye
dye-dependent correction factor at 280 nm (to be delivered from dye-supplier)
Abs.
max, dye
absorbance at absorption maximum of the dye (AU)
A280 factor molecular weight
prot.
/ molar extinction coefficient (M
-1
*cm
-1
)
prot.
or
1 / extinction coefficient (l/g*cm)
lid factor virtual dilution factor 5, 10, 50, 100 and 250; dependent on the used dilution lid.

Calculation of fluorescence dye concentration [pmol/µl]
c
dye
= (Abs.
max, dye
– Abs. 320) * lid factor * dilution factor / (ε
dye
* 10
-6
)
c
dye
dye concentration (pmol/µl)
Abs.
max, dye
absorbance at absorption maximum of the dye (AU)
lid factor virtual dilution factor 5, 10, 50, 100 and 250; dependent on the used dilution lid.
ε
dye
dye-dependent molar extinction coefficient (M
-1
* cm
-1
)