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Healthcare White Paper
Do you know what your Protein
Kinase C is doing?
Elucidating PKC’s elusive functions with ChemGen’s
CordaShield TR-FRET Protein Kinase C assay, using Bjorn
Technologies’ Spectrum plate reader.
Kinase C (PKC) enzymes make up a diverse family of enzymes
defined by the phosphorylation of proteins on serine and
threonine amino acids under specific signaling conditions.
PKCs are involved in a wide array of cellular functions,
including cell growth, cell proliferation, cell movement
and neuronal activity. PKCs are also known to play a role
in several diseases, including Alzheimer’s Disease, cancer,
diabetes and cardiovascular disorders.
the wide spectrum of normal and abnormal cellular functions
in which PKCs have been implicated, it is critical for
researchers to elucidate PKCs precise function. Doing
so, however, has proved difficult.
scientists have successfully cloned ten distinct human
isoforms of PKC. The isoforms’ specific actions in individual
tissue types, however, have yet to be determined. Further,
studies continue to add to the number of PKC-specific
substrates and receptors that activate PKCs. These factors
make the further elucidation of PKC’s function quite challenging.
A TR-FRET screening assay, however, can be an effective
aid for researchers working on a vast array of disorders
and processes in which PKCs play a role.
The CordaShield TR-FRET platform from ChemGen uses terbium
as the donor species and fluorescein as the acceptor group.
The terbium/fluorescein base of CordaShield has several
advantages. Assay optimization is simplified, the kinetics
of complex formation are faster, steric problems are avoided,
as are the cost and lot-to-lot consistency issues encountered
with directly labeled substrates.
terbium-based CordaShield has several unique properties:
Technologies’ Spectrum microplate reader provides an ideal
platform for further simplification of ChemGen’s CordaShield
assay development. Spectrum is a multifunctional plate
reader. It effectively combines the rapid plate reading
technology necessary for high throughput screen with the
enhanced performance and sensitivity needed to read small
fluid volumes. Spectrum has been designed to read all
HTS detection modes— including fluorescence intensity,
time-resolved fluorescence, fluorescence polarization,
luminescence, Alphascreen TM, and absorption—in all plate
formats, up to 1536 wells.
- CordaShield’s ratiometrocity eliminates well-to-well
- CordaShield is time-resolved, permitting the use
of fluorescein with no compound interference.
- Fluorescein simplifies assay development and costs.
Spectrum also uses a customized, application-specific
module in conjunction with an optical reading head. The
head features five photomultiplier tubes that can simultaneously
measure two emission signals at any wavelength. The optical
design provides exceptional sensitivity and accuracy in
fluorescence and luminescence assays, and simultaneous
measurements effectively minimize assay read times.
features provide Spectrum with the speed and sensitivity
necessary to take full advantage of CordaShield’s assay
results demonstrate the acute sensitivity of CordaShield
PKC assays, in that they require sub ng/ml kinase concentrations
thereby reducing overall screening costs. Assays have
also been shown to be robust, with Z’ values exceeding
0.7, thus providing confidence the assay data are meaningful.
Bjorn Technologies has optimized its TR-FRET photomultiplier
tubes to allow for the most sensitive CordaShield detection
system on the market, and designed an optic module specifically
for CordaShield, further simplifying assay setup.
CordaShield TR-FRET Protein Kinase C Assay Using the Spectrum
- A specific optical module for application of ChemGen’s
assay on Bjorn’s Spectrum
- Duel wavelength detection of terbium and fluorescein
- Strong, long-lasting donor signal that minimizes background
- Rapid and reliable detection of PKC activity
Healthcare White Paper
Making the most of your assays, your
cells and your time.
Measuring multiple paramaters within the same well with
Betamer’s Multiplexed Cell Viability and Apoptosis Assays
performed on the Spectrum.
screening facilities today face rising demands to produce
more information from existing compound libraries. One
way to obtain this information is to run sequential assays,
in which the researcher looks at one parameter after another
in separate plates. While this method may produce the
desired data, the time and cost requirements are considerable.
avoid these drawbacks, researchers may opt to generate
data by performing assays in a multiplexed format. Multiplexed
assays allow for multiple parameters to be measured within
the same well. One example of this is to the method of
cell death—apoptosis or necrosis—following a certain treatment
protocol. By performing two assays in a single well, researchers
can obtain information on a drug’s mode of action in less
time, with less consumable cost, and using smaller amounts
of valuable test compounds.
multiplexing can be conducted using multiple cell-based
assays multiplexed together. Cell assays have inherent
properties that make them attractive for multiplexed cell-based
applications. Indeed, cell-based assays are particularly
at risk of variation due to plate-to-plate differences
in cell growth and metabolism. And cell culture is inherently
by using multiplexing assays, researchers can acquire
the same amount of data using fewer cells, and, as the
same cells can be used for subsequent assays, data are
also more precise.
Betamer’s multiplexed cell viability
and apoptosis assays using Bjorn Technologies’ Spectrum
- Correlated measurement of an apoptosis marker and cell
- Spectrum monitors for both luminescence and fluorescence
output signals from the same assay well
- Assay miniaturization up to a 1536-well format
of the experiments shown here demonstrate the ability
to perform different assays within the same assay well.
First using a non-lytic assay, such as Betamer’s [Cell
Titer-Blue], allows for the sequential multiplexing of
several different reagents within the same well. Multiplexed
cell-based assays, with a multifunctional plate reader
that records multiple output signals from the same assay
well, allow researchers to measure multiple parameters
within the same well, thus obtaining precise results faster
and with less consumable usage.
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