NanoPhotometer
®
P-Class
User Manual
Version 2.1
Page 22 / 70
4.2.3 Protein UV Dye Method
The procedure is as follows:
Parameter Screen
NanoVolume Applications
Cuvette Applications
Parameter Screen
Step 1
Press 1 for
NanoVolume
OR 2 for
Cuvette
folder.
Step 2
Press 2 to select
Protein
folder.
Step 3
Press 2 to select
Protein dye
mode.
Step 4
Using NanoVolume Applications select the
Lid Factor
as
described in the “Average Detection Range Sheet” and
under 3.2. A minimum of 1.5 µl sample volume (for lid
10) is recommended.
Step 5
Enter the
Dilution Factor
using the keypad numbers.
Range 1.00 to 9,999. Use the C button to backspace and
clear the last digit entered. OR press
Menu/Options
to
enter the dilution factor screen. Enter the volume of the
sample using the keypad numbers. Range 0.01 to 9,999.
Enter the volume of the diluent using the keypad
numbers. Range 0.01 to 9,999. Press
OK
to calculate
the dilution factor and return to the Parameters screen
OR press
Cancel
to cancel the selections and return to
the Parameters screen.
Step 6
Select whether the
Dye
correction (calculation of the dye-
dependent correction factor) is used or not with the left
and right arrows. The
Background
correction is always
calculated in the Dye methods.
Step 7
Select the
Protein
(BSA (default), Serum Albumin
(mouse), Serum Albumin (human), IgG, Lysozyme,
Custom or OD 1).
Step 8
If using
Custom Protein
there are two possibilities to
enter the correct factors (see also page 18 protein UV
method):
Molar extinction coefficient (M
-1
* cm
-1
):
Ranges are:
Wavelength:
200 nm to 340 nm
Molar extinction coefficient
(M
-1
* cm
-1
)
:
10,000 to
9,999,999
Molecular weight:
0.001 to 9,999,999
Extinction coefficient (l/g * cm):
Ranges are:
Wavelength:
200 nm to 340 nm
Extinction coefficient
(l/g * cm)
:
0.001 to 9,999
Step 9
Select the
Units
of measurement using the left and right
arrows. Options: mg/ml, μg/ml, ng/μl and μg/μl.
Step 10
Enter the protein-dependent extinction coefficient. Range
is 10,000 to 9,999,999.
Step 11
Press
OK
to store the chosen parameters and to enter
the next screen OR
Cancel
to return to the
Protein
folder.
Step 12
Select the appropriate Dye Type. 4 different AlexaFluors,
2 Cy-Dyes, 2 DyLight Dyes, FITC, Pacific Blue, r-PE and
Texas Red are programmed with their corresponding
maximum absorbance wavelength, dye-dependent
extinction co-efficient and dye-dependent correction
factor at 280 nm. For further details please refer to 12.4
Protein fluorescent dye incorporation.
Step 13
If using
Custom Dye
maximum absorbance wavelength of
the custom dye, dye-dependent extinction coefficient and
dye-dependent correction factor at 280 nm have to be
entered. Ranges are:
Dye Abs Max:
300 nm to 950 nm
Dye Ext. Coefficient:
10,000 to 9,999,999
Dye Correction:
0 to 0.999