Operating Procedures
0112-0127 B
105
Using Spectral Scanning to Optimize Excitation and
Emission Wavelengths for Fluorescence Assays
Put 200
μ
L of sample that includes the fluorophore and 200
μ
L of a
buffer control into separate wells of a microplate.
Perform the Excitation Scan
1.
Using the SoftMax Pro Software, set up a Plate section for a
fluorescence read, spectrum mode, Em Fixed/Ex Scan, with no
cutoff filter (default), and medium PMT.
2.
Set the emission wavelength based on the tentative value from
the literature, or from a customary filter set used to measure
your fluorophore. If the emission wavelength is not known,
select a tentative emission wavelength about 50 nanometers
greater than the absorbance maximum of the fluorophore. If
necessary, the absorbance maximum can be determined by
performing an optical density spectral scan first.
3.
Set the excitation scan to start/stop approximately 50 nm
below/above the tentative excitation value obtained from the
literature, or the customary excitation filter.
4.
Set the step increment to 2 nm or 3 nm. You can choose to do a
preliminary scan with a 10 nm increment to determine the
approximate peak location, and then repeat the scan over a
narrower wavelength range with a 2 nm or 3 nm increment.
5.
Perform the scan and view the results as a plot of emission
fluorescence vs. excitation wavelength.
Note the excitation wavelength at the emission peak and the
maximum RFU value.
If an error message reporting missing data points occurs, it might
be due to possible saturation reported by the SoftMax Pro
Software at the end of the spectral scan. Reset the PMT to
Low
and re-scan the sample. Scan the buffer blank with the PMT set
to
Medium
or
High
. If the error occurs after scanning with the
PMT set to
Low
, it might be necessary to dilute the sample.
If the excitation scan shows no apparent peak, change the PMT
setting to
High
and re-scan the sample. If the spectral scan still
shows no apparent peak, adjust the Y-scale of the zoom plot so
that the plot fills the graph.
FlexStation_manual.book Page 105 Tuesday, June 25, 2013 10:22 AM
