Molecular Devices FlexStation II, Руководство оператора

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3 Operating Procedures
1 Preliminary Excitation Scan (Scan Figure 1)
a
Using SOFTmax PRO, set up a Plate section for a fluorescence read, spectrum mode,
Em Fixed/Ex Scan, with no cutoff filter (default), and medium PMT.
b
Set emission wavelength based on the tentative value from the literature (or from a
customary filter set used to measure your fluorophore) and add 30 nm. 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 a spectral scan in a
UV/Vis spectrophotometer.
Scan Figure 1
c
Set excitation scan to start/stop approximately 50 nm below/above the tentative
excitation value obtained from the literature (or the customary excitation filter).
d
Set step increment to 1 or 2 nm. (You may 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 1 or 2 nm increment.)
e
Perform 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.
Note: If an error message reporting missing data points 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: If the excitation scan shows no apparent peak, change the PMT setting to
‘high’ and rescan 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.
f
Select the optimal excitation wavelength. If the excitation peak wavelength and
emission wavelength are separated by more than 30 nm, use the excitation peak
wavelength value. If the excitation and emission wavelengths are less than 30 nm
apart, use the shortest excitation wave-length that gives 90% maximal emission.
80 FlexStation II Operator’s Manual – Rev. D