Molecular Devices SpectraMax Paradigm User Manual page 39

You can use the Protocol Manager in the SoftMax Pro Software to quickly find and open a
predefined protocol.
More protocols and updated protocols can be downloaded from the Knowledge Base on the
Molecular Devices support web site (www.moleculardevices.com/support) or from the
protocol sharing web site (www.softmaxpro.org).
The following detection cartridges have time-resolved fluorescence read mode capability:
Tunable Wavelength (TUNE) Detection Cartridge, see page 97
Multi-Mode (MULTI) Detection Cartridge, see page 102
Cisbio HTRF Detection Cartridge on page 110
Time Resolved Fluorescence (TRF) Detection Cartridge, see page 113
Analyzing Time-Resolved Fluorescence Data
A time-resolved fluorescence (TRF) measurement includes a number of pulses. Each pulse
consists of turning the light source on and off (Excitation Time), pausing for a specified length
of time (Measurement Delay), and measuring the fluorescence intensity of the sample for a
specified length of time (Integration Time). These pulses are repeated several times, as
specified in the protocol parameters.
Analyzing and interpreting TRF data generally consists of the following:
Blank Correction on page 39
Data Normalization on page 40
Data Optimization on page 40
Blank Correction
Although background is significantly lower than with fluorescence intensity measurements,
Molecular Devices recommends that you use blanks or assay controls.
A blank well contains everything used with the sample wells except the label and sample-
specific compounds. Do not use an empty well for a blank.
The blank sample reveals the offset underlying each data sample. This offset does not carry
information on the label, and is generally subtracted before data reduction is done.
For optimum results, Molecular Devices recommends that you run replicates for all blanks,
controls, and samples. In this case, the blank value that can be subtracted is the average
value of all blanks.
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Chapter 2: Read Modes and Read Types
39