LANCE Ultra PAK2 Kinase Assay

Using ULight-Tyrosine Hydroxylase (Ser40) Peptide
& Europium-anti-phospho-Tyrosine Hydroxylase
(Ser40) Antibody
ULight ™-Tyrosine Hydroxylase (Ser40) Peptide:
• TRF0111-D: 0.5 nmole, 1,000 assay points*
• TRF0111-M: 5 nmoles, 10,000 assay points*
*0.5 pmol/assay point
• CORE SEQUENCE MOTIF: RRQSLIE
Synthetic peptide containing the residues
surrounding Ser40 of tyrosine hydroxylase
Phosphorylation site: Ser40
• VALIDATED FOR KINASES: PAK2, Aurora A,
PKA, MSK1, CHK1
Europium-anti-phospho-Tyrosine
Hydroxylase (Ser40) Antibody:
• TRF0204-D: 10 µg, 1,562 assay points*
• TRF0204-M: 100 µg, 15,625 assay points*
*40 fmol/assay point
• RECOGNIZED MOTIF: RRQpSLIE
• Europium-labeled rabbit polyclonal antibody
recognizing phospho-Ser40 in human tyrosine
hydroxylase
LANCE Ultra Kinase Assays
LANCE Ultra time-resolved fluorescence resonance
energy transfer (TR-FRET) assays use a proprietary
europium chelate donor dye, W-1024 (Eu), with
ULight, an innovative small molecular weight
acceptor dye with a red-shifted fluorescent emission.
In kinase assays, the binding of an Eu-labeled antiphospho-substrate antibody to the phosphorylated
ULight-labeled substrate brings donor and acceptor
molecules into close proximity.
After irradiation of the kinase reaction at 320 or 340
nm, the energy from the Eu donor is transferred to the
ULight acceptor dye which, in turn, generates light at
665 nm. The intensity of the light emission is proportional to the level of ULight-substrate phosphorylation.
Development of a PAK2 Kinase Assay
Additional Reagents
PAK2, active
Upstate # 14-481
LANCE Detection
Buffer, 10X
PerkinElmer # CR97-100
OptiPlate™-384, white
PerkinElmer # 6007299
TopSeal-A™
PerkinElmer # 6005185
Kinase Buffer: 50 mM HEPES, pH 7.5, 1 mM EGTA,
10 mM MgCl2, 2 mM DTT and 0.01% Tween-20
w w w. p e r k i n e l m e r. c o m
N O T E
Two LANCE® Ultra companion products —
two convenient sizes!
T E C H N I C A L
LANCE Ultra PAK2 Kinase Assay
L A N C E U LT R A T R - F R E T T E C H N O L O G Y
TECH NOTE U-TRF #8
Suggested Procedure
• Dilute kinase, ATP, inhibitors and
ULight-Tyrosine Hydroxylase
peptide in Kinase Buffer.
Experiment 1:
Enzymatic Time Course
Experiment 2:
ATP Titration
PAK2 enzyme was incubated at concentrations
ranging from 0.125 to 4 nM with 50 nM ULightTyrosine Hydroxylase peptide and 20 µM ATP.
Kinase reactions were terminated after 0 to 120
min by the addition of EDTA.
Serial dilutions of ATP ranging from 10 nM to
1 mM were added to 0.5 nM PAK2 and 50 nM
of ULight-Tyrosine Hydroxylase peptide.
Kinase reactions were terminated after 30 min
by the addition of EDTA.
Experiment 3:
Enzyme Inhibition Curve
Experiment 4:
Z'-factor Determination
Serial dilutions of staurosporine ranging from
3 pM to 30 µM (final concentrations in 2%
DMSO) were incubated with 0.5 nM PAK2,
50 nM ULight-Tyrosine Hydroxylase peptide
and 10 µM ATP. Kinase reactions were
terminated after 30 min by the addition of EDTA.
PAK2 enzyme at 0.5 nM was incubated with
50 nM ULight-Tyrosine Hydroxylase peptide
and 10 µM ATP with or without 10 µM
staurosporine (final concentrations in 2%
DMSO). Kinase reactions were terminated
after 30 min by the addition of EDTA.
• Prepare a 4X Detection Mix by
diluting the Eu-anti-phosphoTyrosine Hydroxylase antibody to
8 nM in 1X LANCE Detection Buffer.
• Add to the wells of a white
OptiPlate-384:
5 µL of PAK2 enzyme,
2.5 µL of inhibitor or Kinase
Buffer,
2.5 µL of ULight-Tyrosine
Hydroxylase Peptide/ATP mix
(for ATP titration, ATP dilutions
are added separately in Kinase
Buffer).
• Cover the plate with TopSeal-A and
incubate at room temperature (RT).
• Stop the kinase reactions by adding
5 µL of 40 mM EDTA prepared in
Detection Buffer. Leave for 5 min
at RT.
• Add 5 µL of Detection Mix (Euanti-phospho-Tyrosine Hydroxylase
antibody at a final concentration
of 2 nM).
• Cover with TopSeal-A and incubate
for 1 h at RT.
• Remove TopSeal-A and read signal
with the EnVision™ Multilabel
Reader in TR-FRET mode (excitation
at 320 nm and emission at 665 nm).
NOTE: Eu-labeled antibodies and EDTA can
be premixed before use as a 2X concentrated
Stop/Detection mix to minimize the number
of liquid handling steps.
Better PAK2 Kinase Assays with a Better Technology—
LANCE Ultra
For more information about LANCE Ultra, please visit
www.perkinelmer.com/lanceultra or contact your local
PerkinElmer Sales Representative. Learn more about our
comprehensive range of reagents and consumables for drug
discovery by visiting www.perkinelmer.com/drug discovery.
PerkinElmer Life and
Analytical Sciences
710 Bridgeport Avenue
Shelton, CT 06484-4794 USA
Phone: (800) 762-4000 or
(+1) 203-925-4602
www.perkinelmer.com
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©2006 PerkinElmer, Inc. All rights reserved. The PerkinElmer logo and design are registered trademarks of PerkinElmer, Inc. LANCE is a registered trademark and EnVision, OptiPlate,
TopSeal-A and ULight are trademarks of PerkinElmer, Inc. or its subsidiaries, in the United States and other countries. ULight is covered under patent number US2005202565 and
US20040166515. All other trademarks not owned by PerkinElmer, Inc. or its subsidiaries that are depicted herein are the property of their respective owners. PerkinElmer reserves the
right to change this document at any time without notice and disclaims liability for editorial, pictorial or typographical errors.
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