Redder is Better: Far-Red PolarScreen™
FP Kinase Assays
Kevin L. Vedvik, Hildegard C. Eliason, Jennifer A. Fronczak,
Melissa E. Krueger and Kurt W. Vogel
Invitrogen Corporation • 501 Charmany Drive • Madison, WI 53719 • USA
Why is Redder Better?
Compound interference is the bane of HTS. Two forms of interference that affect fluorescence polarization assays are compound fluorescence and
light scatter. Compound fluorescence arises from the intrinsic fluorescence of some library compounds. Because compound libraries are typically
screened at concentrations of 10 µM, and tracer concentrations are typically 1 nM, a library compound that has 1/10,000th (or less) of the fluorescent “brightness” of the tracer has the potential to interfere with the assay signal. Because most small molecules that fluoresce will emit a depolarized (low polarization) signal, this can lead to false positive or false negative results, depending on the assay configuration.
The ability of a molecule to absorb light in the “red” (longer wavelength) region of the spectrum depends largely on the
degree of conjugation (adjacent double bonds and aromatic rings) within the molecule. In general, the more conjugation the
molecule has, the better able it will be to absorb light in the red region of the spectrum. Because the ability to absorb light is
a prerequisite to fluorescence, it follows that in order to fluoresce at “redder” regions of the spectrum a molecule needs more
conjugated double bonds. The high degree of conjugation necessary for “red” fluorescence is incompatible with “druglike”
properties, and therefore there are, in general, fewer “red” fluorescent compounds in most libraries.
The second interfering factor in FP assays arises from light scatter. Light scatter arises from precipitated compounds or other particulate matter
(dust) in an assay well. Because light scatter is more efficient at lower (greener) wavelengths, and drops off in intensity relative to the 4th power of
the wavelength, scatter is less of an interference at longer wavelengths. Scattered light is often polarized (this is the reason sunglasses are polarized—to block scattered light, or glare, from the horizon) and therefore interferes with an assay by giving an abnormally high polarization value.
Invitrogen Corp • 1600 Faraday Avenue • Carlsbad, CA 92008 USA • Tel: 760 603 7200 • FAX: 760 602 6500 • E-mail: [email protected] • www.invitrogen.com
Figure 1—Assay Interference Decreases at Longer Wavelengths
Compounds in LOPAC1280 with greater than 50% signal intensity of 1 nM corresponding tracer
Tracer
λ (Ex / Em)
# of Compounds
Green
485 / 535
19
Red
535 / 590
9
Far-Red
590 / 650
3
To determine an approximation of the amount of fluorescent interference that can be expected in a typical compound library at green,
red, and far-red wavelengths, the LOPAC1280 library was prepared at 10 µM in buffer and read in fluorescence intensity mode using filters
typical to a green, red, or far-red tracer. Wells containing 1 nM of each tracer were placed on the assay plate so that signal intensity
could be referenced to a typical tracer concentration.
3.0
Relative Interference from Scatter
Relative Signal Intensity
2.5
2.0
1.5
GW5047
1.0
0.5
0.0
Green Tracer
Green Scatter
Red Tracer
Red Scatter
Far Red Tracer Far Red Scatter
To demonstrate the effect of light scatter on assay interference, GW5047, a compound from the LOPAC Library prone to precipitation,
was compared in signal intensity to 1 nM of green, red, and far-red tracers. The graph above illustrates negligible interference (<
0.4%) seen from GW5047 when using a far-red tracer. The inset shows a close-up of a vial containing the precipitated compound.
Figure 2—Excitation / Emission Spectra of Far-Red Tracers
Fluorescein
Rhodamine
Far Red
100
75
50
25
0
400
450
500
550
600
650
700
750
Wavelength (nm)
Excitation/Emission spectra of commonly-used fluorescein and rhodamine-based tracers compared to the spectra of Invitrogen’s new
Far-Red tracers. The excitation and emission maxima for the far-red tracer are red-shifted beyond 600 nm, allowing fluorescence polarization assays to be performed using a 610 nm excitation filter and 670 nm emission filters.
Figure 3—FP Kinase Assay Schematic
Add Antibody
and Tracer
ATP
P
ANTIBODY
P
ANTIBODY
P
Kinase
Detection
P
P
P
P
P
ANTIBODY
Kinase
ATP
+
Inhibitor
P
P
Tracer
High Polarization
ANTIBODY
Tracer
Low Polarization
Inhibited Reaction Uninhibited Reaction
Kinase Reaction
In a far-red FP kinase assay, kinase, substrate, and ATP are allowed to react in the presence of library compounds. After the reaction,
antibody and far-red-labeled tracer are added. The amount of antibody that binds to the tracer is inversely related to the amount of phosphorylated product present. Thus, library compounds that inhibit the reaction are identified as wells that have a high polarization value.
Figure 4—Far Red FP Kinase Kits Address a Range of Kinase Targets
RbING Far-Red Assay (Kinase: CDK5/p35)
PTK Far-Red Assay (Kinase: LynB)
250
250
IκB-α pSer 32 Far-Red Assay (Kinase: CKII)
300
200
175
150
125
200
250
Polarization (mP)
Polarization (mP)
Polarization (mP)
225
200
150
150
100
100
50
10-5
10-4
10-3
10-2
10-1
100
101
10-5
10-4
[Kinase] (ng/well)
10-3
10-2
10-1
100
101
10-5
PKC Far-Red Assay (Kinase: CHK1)
10-3
10-2
10-1
100
101
[Kinase] (units/well)
PDK1 Far-Red Assay (Kinase: PDK1)
225
Crosstide Far-Red Assay (Kinase: Akt1/PKBα)
200
200
175
150
125
150
Polarization (mP)
Polarization (mP)
200
Polarization (mP)
10-4
[Kinase] (ng/well)
100
50
150
100
100
75
0
10-5
10-4
10-3
10-2
10-1
100
50
10-4
10-3
[Kinase] (ng/well)
10-2
10-1
100
101
[Kinase] (pg/well)
10-5
10-4
10-3
10-2
10-1
100
[Kinase] (ng/well)
CREBtide Far-Red Assay (Kinase: PKA)
250
Polarization (mP)
225
200
175
150
125
100
10-2
10-1
100
101
102
103
[Kinase] (pg/well)
Seven far-red kinase assays are available to address a wide variety of kinase targets. The plots in Figure 3 show the results from kinase
titrations in 10 µL, 90 minute kinase reactions under conditions of non-limiting substrate and ATP. In general, picogram to nanogram
quantities of kinase will phosphorylate sufficient peptide substrate to effect an assay window of between 125 and 250 mP.
Figure 5—Inhibition of PDK1 by Staurosporine
Inhibition of PDK1 with Staurosporine
175
EC50 = 9.4 nM
Polarization (mP)
150
125
100
75
50
25
0
0.1
1
10
100
[Staurosporine] (nM)
Far-Red kinase assays can be used for both high-throughput screening as well as follow-up of hits to determine accurate EC50 values.
Conclusions
As the size of compound libraries increases so does the cost of follow-up screening of false positive and false negative “hits” that are
due to compound interference. The shift from “green” to “red” has been recognized as a valid strategy to overcome interference due to
autofluorescence or light scatter due to precipitated compounds. Invitrogen’s new far-red PolarScreen™ assays employ a proprietary farred fluorophore that gives excellent performance in fluorescence polarization assays. The fluorophore is highly water soluble and unlike
cyanine-based fluorophores has a fluorescence lifetime that allows for large polarization shifts between free and bound tracer.
839-0412392 041504