NanoPhotometer
®
NP80/N60/N50/C40
User Guide Version 3.1
60
DOL
Degree of labeling/dye per protein ratio
A
max,dye
Absorbance value at the absorbance maximum of the dye (10 mm path)
A
280
Absorbance at 280 nm (10 mm path)
A
BKG
Absorbance at selected background wavelength (10 mm path)
ε
dye
Extinction coefficient of dye in M
-1
*cm-
1
ε
prot
Extinction coefficient/protein factor in g*cm/l
cf
dye
Dye-dependent correction factor at 280 nm
Table 4. Dye Types, Absorbance Max, Extinction coefficients, and dye-dependent
correction factors
NanoPhotometer
®
Models
Dye Type
Absorbance
maximum
of Dye (nm)
Dye-dependent
ext. coeff.
ε
dye
in M
-1
*cm
-1
Dye-dependent
correction
factor at 280 nm
cf
dye
NP80/N60/N50/C40 Alexa Fluor 350
346
19,000
0.19
NP80/N60/N50/C40 Alexa Fluor 405
401
34,500
0.70
NP80/N60/N50/C40 Alexa Fluor 488
494
71,000
0.11
NP80/N60/N50/C40 Alexa Fluor 532
530
81,000
0.09
NP80/N60/N50/C40 Alexa Fluor 546
554
104,000
0.12
NP80/N60/N50/C40 Alexa Fluor 555
555
150,000
0.08
NP80/N60/N50/C40 Alexa Fluor 568
577
91,300
0.46
NP80/N60/N50/C40 Alexa Fluor 594
590
73,000
0.56
NP80/N60/C40
Alexa Fluor 647
650
239,000
0.03
NP80/N60/C40
Alexa Fluor 680
679
184,000
0.05
NP80/N60/C40
Alexa Fluor 790
785
260,000
0.08
NP80/N60/N50/C40 Cy3
550
150,000
0.05
NP80/N60/C40
Cy5
649
250,000
0.05
NP80/N60/C40
DyLight 649
654
250,000
0.04
NP80/N60/N50/C40 DyLight 488
493
70,000
0.15
NP80/N60/N50/C40 FITC
495
68,000
0.30
NP80/N60/N50/C40 Pacific Blue
409
30,000
0.20
NP80/N60/N50/C40 Pacific Orange
397
24,500
0.60
NP80/N60/N50/C40 pHrodo Green
505
75,000
0.20
NP80/N60/N50/C40 pHrodo Red
560
65,000
0.12
NP80/N60/N50/C40 r-PE
566
200,000
0.18
NP80/N60/N50/C40 Texas Red
595
80,000
0.18
Ratios
Protein samples e.g. from whole cell lysates may contain nucleic acids. To check the purity of
the isolated protein, the 260/280 ratio is calculated to give an indication of the nucleic acid
contamination. A pure protein preparation has an expected 260/280 ratios of
∼
0.57. If a ratio is
