Implen P 330 - Protein Fluorescent Dye Incorporation

NanoPhotometer
®
P-Class User Manual
Version 2.1 Page 70 / 70
 Calculation of degree of labelling (D/P)
degree of labelling = (Abs.
max, dye
– Abs. 320) * ε
prot
/ [(Abs. 280 - Abs.320) * ε dye]
With dye correction:
degree of labelling = (Abs.
max, dye
– Abs. 320)*ε
prot
/ [(Abs. 280 - Abs.320– ((Abs.
max, dye
– Abs. 320) * CF
dye
)) * ε dye]
Abs.
max, dye
absorbance at absorption maximum of the dye (AU)
ε
prot
protein-dependent molar extinction coefficient (M
-1
* cm
-1
)
CF
dye
dye-dependent correction factor at 280 nm (to be delivered from dye-supplier)
ε dye dye-dependent molar extinction coefficient (M
-1
* cm
-1
)
The following dye types and parameters are pre-programmed in the NanoPhotometer
®
P-Class.
Dye Type
Absorption maximum
Dyes (nm)
Dye-dependent extinction
coefficient ε
dye
Dye–dependent correction
factor (280 nm) CF
Dye
Alexa Fluor 350
346
19,000
0.19
Alexa Fluor 405
401
34,500
0.70
Alexa Fluor 488
495
71,000
0.11
Alexa Fluor 647
650
239,000
0.03
Cy3
550
150,000
0.05
Cy5
649
250,000
0.05
DyLight 649
654
250,000
0.04
DyLight 488
493
70,000
0.15
FITC
495
68,000
0.30
Pacific Blue
416
46,000
0.20
r-PE
566
200,000
0.18
Texas Red
595
80,000
0.18
*In all formulas the molar dye-dependent and the molar protein-dependent extinction coefficient is used.