User manual

Section 3- Applications
- uM - is the concentration based upon the respective dye’s extinction coefficient. Concentration units may
be selected from the adjacent drop-down list box.
• A280 - displays the absorbance at 280 nm for the protein sample measured. The displayed absorbance
value is normalized to a 10 mm path.
Note: The displayed A280 value is the difference in absorbance of the sample at 280 nm compared to
the absorbance of the sample at 750 nm. The A280 value used to calculate the protein
concentration takes into account any appropriate dye correction factors, the absorbance
correction due to the selected analysis correction nm as well as the 750 nm baseline. Therefore,
the displayed A280 value may not be the value used to calculate the protein concentration.
• Analysis correction -the absorbance at the specified correction wavelength is subtracted from the
absorbance at the analysis wavelength before calculating the concentration. This only affects the reported
protein concentration.
• Sloping Dye Correction - when selected the software will subtract the value of a sloping baseline from 400
to 750 nm from the absorbance at the Dye wavelength. This only affects the reported absorbance of the
dye peak(s) and dye concentration(s).
Making Proteins & Labels Measurements
1. Select the Proteins & Labels 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 concentration box. The
default units are mg/mL.
4. Use the drop-down lists in the Dye 1 or Dye 2 box to select the appropriate dye (or dyes). The default for
Dye 1 is Cy3 and the default for Dye 2 is Cy5. If the protein has been labeled with only one dye choose
None as the dye type for Dye 2.
5. A default wavelength of 340 nm is automatically used for a bichromatic normalization for the protein
component of the sample. Select an alternative reference wavelength or choose not to have the spectrum
normalized by deselecting Analysis correction.
- Select the file drop-down option Use current settings as default as a convenient way to limit set-up time
for each new workbook.
6. 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.
7. Select Overlay spectra to display multiple spectra at a time.
8. Select Sloping dye correction 400-750 nm if desired.
9. Establish a blank using the appropriate buffer. The reference or 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 dH
2
O onto the bottom pedestal, lower the arm and click the Blank
button.
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