2. General Description
2016-04
IFU for Infinite 200 PRO No. 30052730 Rev. No. 1.6
23
2.4.3
Luminescence
Glow Type Chemi- or Bioluminescence
The Infinite 200 PRO provides measurement of glow type chemi- or
bioluminescence. Glow type means that the luminescence assay glows much
longer than a minute. Luminescence substrates are available, which provide
stable enough light output over hours.
As an example, luminescence can be measured to determine the activity of an
enzyme labeled compound (-peroxidase, -phosphatase). Light emission results
from a luminescence substrate being decomposed by the enzyme. Under excess
of substrate the luminescence signal can be assumed to be proportional to the
abundance of the enzyme labeled compound. As with enzyme-based assays,
control of environmental conditions is rather critical (temperature, pH-value).
For practical aspects of luminescence assays see the following example:
Bioluminescence Methods and Protocols,
ed. R.A. LaRossa, Methods in
Molecular Biology 102, Humana Press, 1998.
Bioluminescence Resonance Energy Transfer (BRET)
BRET is an advanced, non-destructive, cell-based assay technology that is
perfectly suited for proteomics applications, including receptor research and the
mapping of signal transduction pathways. BRET is based on energy transfer
between fusion proteins containing
Renilla luciferase
(Rluc) and a mutant of the
Green Fluorescent Protein (GFP). The BRET
signal is generated by the oxidation
of p.a. DeepBlueC, a coelenterazine derivative that maximizes spectral resolution
for superior sensitivity. This homogeneous assay technology provides a simple,
robust and versatile platform with applications in basic academic as well as
applied research.
Flash Luminescence
In flash type luminescence assays, the measurement is only done during the
dispensing of the activating reagent or after a short delay time (for Flash
luminescence measurements with the Infinite 200 PRO, see also 2.3.1 Injector
Measurement).
Over the past years luminescence substrates have been improved towards
providing more stable signals. In so-called glow type luminescence assays the
luminescence signal is spread over a wide time scale (e.g. a half-life of 30 min.).
2.4.4
AlphaScreen/AlphaLISA
The Infinite F200 PRO is able to measure Amplified Luminescent Proximity
Homogeneous Assays (AlphaScreen and AlphaLISA). Due to their
nonradioactive, homogenous and sensitive nature, these bead-based
technologies are perfectly suited for the study of biomolecular interactions.
Upon illumination at 680 nm, the photosensitive molecules contained in the donor
beads produce high levels of oxyradicals. These oxyradicals are able to travel to
the acceptor beads and trigger a cascade of reactions that ultimately lead to the
generation of a strong chemiluminescent signal.
