LANCE Ultra p60c-src Kinase Assay - Using ULight - Poly GT (4:1) & Europium-Anti-Phospho-Tyrosine (PY20) Antibody

Using ULight - Poly GT (4:1) &
Europium-Anti-Phospho-Tyrosine (PY20) Antibody
Two LANCE® Ultra companion products —
two convenient sizes!
• TRF0100-D: 1 nmole, 1,000 assay points*
• TRF0100-M: 10 nmoles, 10,000 assay points*
*1 pmol/assay point
• PEPTIDE SEQUENCE: [Glu-Tyr (4:1)] n
• AD0066: 50 µg, 7,800 assay points*
• AD0067: 1 mg, 150,000 assay points*
*40 fmol/assay point
• RECOGNIZED MOTIF: pTyr
• Mouse monoclonal IgG2b antibody directed
against phospho-tyrosine.
Generic substrate for tyrosine kinases
• VALIDATED FOR KINASES: p60c-src, AXL
LANCE Ultra Kinase Assays
• POTENTIAL SUBSTRATE FOR KINASES:
most tyrosine kinases
LANCE® Ultra time-resolved fluorescence resonance
energy transfer (TR-FRET) assays use a proprietary
europium chelate donor dye, W-1024 (Eu), with
ULight, a new 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 phosphorylated
ULight-labeled substrates brings donor and acceptor
molecules into close proximity.
After irradiation of the kinase reaction at 320 nm, the
energy from the Eu donor is transferred to the ULight
acceptor 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 p60c-src Kinase Assay
Additional Reagents
p60c-src, recombinant
Upstate # 14-326
LANCE Detection Buffer,
10X
PerkinElmer
# CR97-100
OptiPlate™-384, white
PerkinElmer
# 6007299
TopSeal-A™
PerkinElmer
# 6005185
Kinase Buffer: 50 mM Tris-HCl, 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
ULight ™-poly GT (4:1):
Europium-anti-phospho-tyrosine (PY20)
antibody:
T E C H N I C A L
LANCE Ultra p60c-src 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 #3
Suggested Procedure
• Dilute kinase, ATP, inhibitors and
ULight-poly GT (4:1) in Kinase
Buffer.
Experiment 1: Enzymatic
Titration Assay
Experiment 2: ATP Titration
p60c-src enzyme at concentrations ranging from
0.3 pM to 30 nM was incubated with 100 nM
ULight-poly GT (4:1) and 20 µM ATP. Kinase
reactions were terminated after 60 min by the
addition of EDTA.
Serial dilutions of ATP ranging from 1 nM to
100 µM were added to 300 pM p60c-src kinase
and 100 nM of ULight-poly GT (4:1) substrate.
Kinase reactions were terminated after 60 min
by the addition of EDTA.
Experiment 3: Enzyme
Inhibition Curve
Experiment 4: Z'-factor
Determination:
Serial dilutions of staurosporine ranging from
100 pM to 10 µM (final concentrations in 2%
DMSO) were pre-incubated for 5 min with the
p60c-src enzyme (300 pM final concentration).
Then 100 nM ULight-Poly GT (4:1) and 4 µM
ATP were added. Kinase reactions were
terminated after 60 min by the addition
of EDTA.
The p60c-src enzyme at 300 pM was incubated
with 100 nM ULight-poly GT (4:1) substrate in
Kinase Assay Buffer with 4 µM ATP or 10 µM
staurosporine and ATP. Reactions were
terminated after 60 min by the addition
of EDTA.
• Dilute antibody (Ab) in LANCE
Detection Buffer to 8 nM.
• Add to the wells of a white
OptiPlate-384:
5 µL of p60 c-src enzyme,
2.5 µL of inhibitor or Kinase
Buffer,
2.5 µL of ULight-poly GT
(4:1)/ATP mix (for ATP titration,
ATP dilutions are added separately
in Kinase Buffer).
• Incubate the enzymatic reaction
at room temperature (RT).
• Stop the reaction by adding 5 µL
of 40 mM EDTA in Detection
Buffer. Leave 5 min at RT.
• Add 5 µL of the antibody dilution
(2 nM final concentration).
• 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).
Better p60c-src 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/
drugdiscovery.
Complementary Products
Product
Description
Product No.
Europium-anti-phospho-Tyrosine (PT66)
50 µg
1 mg
AD0068
AD0069
Europium-anti-phospho-Tyrosine (P-Tyr-100)
10 µg
50 µg
1 mg
AD0203
AD0161
AD0162
ULight-poly GAT, [EAY(1:1:1)] n
1 nmole
10 nmoles
TRF0101-D
TRF0101-M
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
For a complete listing of our global offices, visit www.perkinelmer.com/lasoffices
©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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