User manual

Section 3- Applications
• Ext. Coeff, E1% L/gm-cm - appears when Other Protein (E1%) is selected. The appropriate extinction
coefficient should be entered prior to making a measurement.
•
ε /1000 and M.W. (KDa) - appears when Other protein (E & MW) is selected. The appropriate e/1000 and
M.W. (kDa) should be entered prior to making a measurement.
• Conc. - concentration based on absorbance at 280 nm and the selected extinction coefficient.
Concentration units are chosen from the adjacent drop down list. The default selection is mg/mL.
• A280 (10 mm path) -
absorbance at 280 nm for the protein sample being measured. The displayed
absorbance value is normalized to a 10 mm path.
• 260/280- ratio of the absorbance at 260 and 280 nm.
• Baseline correction: if selected, the default wavelength for the bichromatic normalization is 340 nm. The
user can manually enter a different wavelength for the bichromatic normalization of the absorbance data. In
either case, the baseline is automatically set to the absorbance value of the sample at the selected
wavelength. All wavelength data will be referenced off of this value. If the baseline correction is not utilized,
the spectra may be offset from the baseline and the calculated protein concentration may be higher than the
true value.
Making Protein A280 Measurements
1. Select the Protein A280 application from the main menu. If the wavelength verification window appears,
ensure the arm is down and click OK.
2. Select the type of sample to be measured from the Type drop-down list. The default setting is 1 Abs = 1
mg/mL.
3. Choose the concentration units from the drop-down list adjacent to the color coded box. Default is mg/mL.
4. A default wavelength of 340 nm is automatically used for a bichromatic normalization. Select an alternative
reference wavelength or choose not to have the spectrum normalized by deselecting the Baseline
correction box.
- Select the file drop-down option Use current settings as default as a convenient way to limit set-up time
for each new workbook.
5. Select Add to report to automatically include all measurements in the current report. The default setting is
for all samples to be added to reports. The Add to report checkbox must be selected prior to a
measurement to save the sample data to a workbook.
6. Select Overlay spectra to display multiple spectra at a time.
7. Establish a blank using the appropriate buffer. The blank solution generally is the buffer that the molecule of
interest is suspended or dissolved in. This solution should be the same pH and of a similar ionic strength as
the sample solution.
- Pedestal Option: Pipette 2 µL of the appropriate blanking solution onto the bottom pedestal, lower the arm
and click the Blank button.
- Cuvette Option (Model 2000c only): Insert the cuvette noting the direction of the light path indicated by
the etched arrow. The optical beam (2 mm) is directed 8.5 mm above the bottom of the cuvette. Refer to
the cuvette manufacturer for volume recommendations.
Note: The arm must be down for all measurements, including those made with cuvettes. It is
recommended that cuvettes be removed from the instrument prior to making a pedestal
measurement to ensure that the pedestal arm can move to the proper starting position.
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