TCH_LANCEUltrap60csrcPolyGATPY20

Using ULight-Poly GAT (1:1:1) &
Europium-Anti-Phospho-Tyrosine (PY20) Antibody
ULight ™-poly GAT (1:1:1):
• TRF0101-D: 1 nmole, 1,000 assay points*
• TRF0101-M: 10 nmoles, 10,000 assay points*
*1 pmol/assay point
• PEPTIDE SEQUENCE: [Glu-Ala-Tyr (1:1:1)]n
Europium-anti-phospho-tyrosine
(PY20) Antibody:
• 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 KINASE: p60c-src
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, 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 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
Two LANCE® Ultra companion products —
two convenient sizes!
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 #10
Suggested Procedure
• Dilute kinase, ATP, inhibitors and
ULight-poly GAT (1:1:1) 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 GAT (1:1:1) and 20 µM ATP. Kinase
reactions were terminated after 60 min by the
addition of EDTA.
Serial dilutions of ATP ranging from 10 nM to
1 mM were added to 1 nM p60c-src kinase and
100 nM of ULight-poly GAT (1:1:1) 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 (1 nM final concentration).
Then 100 nM ULight-Poly GAT (1:1:1) and 1.5
µM ATP were added. Kinase reactions were
terminated after 60 min by the addition of EDTA.
p60c-src enzyme at 1 nM was incubated with
100 nM ULight-poly GAT (1:1:1) substrate in
kinase assay buffer with 1.5 µM ATP, 10 µM
staurosporine and ATP, or without 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 p60c-src enzyme,
2.5 µL of inhibitor, or Kinase
Buffer,
2.5 µL of ULight-poly GAT
(1:1:1)/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.
• 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/drug discovery.
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 GT (4:1)
1 nmole
10 nmoles
TRF0100-D
TRF0100-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
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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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Printed in USA