Glossary - Molecular Devices SpectraMax Paradigm User Manual

Glossary

A
Absorbance
Absorbance is the quantity of light absorbed by a solution. To measure absorbance
accurately, it is necessary to eliminate light scatter. If there is no turbidity, then
absorbance = optical density.
A = log
where I
through the sample, and A is the measured absorbance.
See Absorbance Read Mode on page
AlphaScreen
ALPHA stands for Amplified Luminescent Proximity Homogeneous Assay. AlphaScreen® is a
bead-based chemistry used to study molecular interactions between moieties A and B, for
example. When a biological interaction between A and B moves beads (coated with A and B,
respectively) together, a cascade of chemical reactions produce a greatly amplified signal.
The cascade finally resulting in signal is triggered by laser excitation (680 nm), making a
photosensitizer on the A-beads convert oxygen to an excited (singlet) state. That energized
oxygen diffuses away from the A-bead. When reaching the B-bead in close proximity, it reacts
with a thioxene derivative on the B-bead generating chemiluminescence at 370 nm. Energy
transfer to a fluorescent dye on the same bead shifts the emission wavelength into the
520 nm to 620 nm range. The limited lifetime of singlet oxygen in solvent (~4 microseconds)
lets diffusion reach up to only around 200 nm distance. Thus, only B-beads in the proximity
of A-beads yield signal, which indicates binding between moieties A and B.
An AlphaScreen measurement includes a light pulse, by turning on the laser diode for a
specified time, turning off the laser diode, followed by the measurement of the AlphaScreen
signal, as specified in the measurement protocol timing parameters.
Note: AlphaScreen beads are light sensitive. Beads are best handled under subdued
(<100 lux) or green filtered (Roscolux filters #389 from Rosco, or equivalent) light
conditions. Do incubation steps in the dark.
See AlphaScreen Read Mode on page
5014038 E
(I /I) = –log (I/I )
10 0 10 0
is incident light before it enters the sample, I is the intensity of light after it passes
0
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