Molecular Devices SpectraMax GEMINI EM, Operator'S Manual

Page: 39 / 64

Operation
SpectraMax GEMINI EM Operator’s Manual 3-5
centration samples), medium, or high (for lower concentration samples) in
all read modes. In endpoint and spectrum mode, there is an additional set-
ting, automatic, in which the instrument will automatically adjust the PMT
voltage for varying concentrations of sample in the plate.
• The chamber of the SpectraMax GEMINI EM is isothermal at ambient as
well as at elevated temperatures. The temperature in the reading chamber
may be adjusted from 4°C above ambient to 45°C.
Other important factors that are independent of the instrument but which
affect assay optimization include the Stokes shift. When the Stokes’ shift is
very small, optimizing the excitation and emission wavelengths and correct
cutoff
fi lter choices are very important.
Using Spectral Scanning to Optimize Excitation and
Emission Wavelengths for Fluorescence Assays
1) Put 200 µL of sample that includes the fl uorophore and 200 µL of a buffer
control into separate wells of a microplate.
2) Excitation Scan
A) Using SoftMax Pro, set up a plate section for a fl uorescence read, spec-
trum mode, Em Fixed/Ex Scan, with no cutoff fi lter (default), and
medium PMT.
B) Set the emission wavelength based on the tentative value from the lit-
erature (or from a customary fi lter set used to measure your fl uoro-
phore). If the emission wavelength is not known, select a tentative
emission wavelength about 50 nanometers greater than the absor-
bance maximum of the
fl uorophore. If necessary, the absorbance max-
imum can be determined by performing a spectral scan in a UV/Vis
spectrophotometer.
C) Set the excitation scan to start/stop approximately 50 nm below/
above the tentative excitation value obtained from the literature
(or the customary excitation
fi lter).
D) Set the step increment to 1 or 2 nm. (You may choose to do a prelimi-
nary scan with a 10-nm increment to determine the approximate peak
location, and then repeat the scan over a narrower wavelength range
with a 1- or 2-nm increment.)
E) Perform the scan and view the results as a plot of emission fl uores-
cence vs. excitation wavelength. Note the excitation wavelength at the
emission peak and the maximum RFU value.
If an error message reporting missing datapoints occurs, it may be
due to possible saturation reported by SoftMax Pro at the end of the
spectral scan. Reset the PMT to “low” and rescan 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 may be necessary
to dilute the sample.
NOTE: