TCH_LANCEUltraPKCKinaseAssay

ULight-PKC Peptide and Europium-anti-phospho-PKC
(Ala25Ser) Peptide Antibody
Two LANCE Ultra companion products – two convenient sizes!
• TRF0108-D: 0.5 nmole, 1,000* assay points
Europium-anti-phospho-PKC (Ala25Ser)
Peptide Antibody:
• TRF0108-M: 5 nmoles, 10,000* assay points
• TRF0207-D: 10 µg, 1,562* assay points
*0.5 pmol/assay point
• TRF0207-M: 100 µg, 15,625* assay points
PEPTIDE SEQUENCE:
*40 fmol/assay point
CRFARKGSLRQKNV
Synthetic peptide derived from amino acids 19-31 of PKC␣;
phosphorylation site: Ser25.
VALIDATED FOR KINASES:
PKC␣, PKC␤1, PKC␤2, PKC␦, PKC⑀, PKC␥, PKC␩, PKC␫,
PKC␪, PKC␨
RECOGNIZED MOTIF:
RFARKGpSLRQKNV
Europium-labeled mouse monoclonal antibody recognizing
phospho-Ser25 in human PKC in which Ala25 is mutated to
Ser and phosphorylated.
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 a Eu-labeled antiphospho-substrate antibody to the phosphorylated ULightlabeled 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 ULightsubstrate phosphorylation.
Development of a PKC Kinase Assay
Additional reagents
PKC-␣
Invitrogen # P2227
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
ULight™-PKC peptide:
T E C H N I C A L
PKC Kinase Assay
L A N C E U LT R A
TECH NOTE U-TRF #11
Suggested procedure
• Dilute the PKC-␣ kinase, ATP, inhibitors and ULight-PKC
Peptide in Kinase Buffer.
• Stop kinase reactions by adding 5 µL of 40 mM EDTA
prepared in 1X Detection Buffer (Stop Solution). Leave for
5 min at RT.
• Prepare a 4X Detection Mix by diluting the Eu-anti-phospho PKC
(Ala25Ser) antibody to 8 nM in 1X LANCE Detection Buffer.
• Add 5 µL of 4X Detection Mix (Eu-anti-phospho-PKC
(Ala25Ser) Antibody at a final concentration of 2 nM).
• Add to the wells of a white OptiPlate-384:
– 5 µL of PKC-␣ enzyme
– 2.5 µL of inhibitor or Kinase Buffer
– 2.5 µL of ULight-PKC substrate/ ATP mix (for ATP titration,
ATP dilutions are added separately in Kinase Buffer).
• Cover with TopSeal-A and incubate for 1 h at RT.
• Cover the plate with TopSeal-A and incubate for
30 min at room temperature (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 Solution/Detection Mix to minimize
the number of liquid handling steps.
Experiment 1: Enzymatic Time Course
Experiment 2: ATP Titration
PKC-␣ enzyme was incubated at concentrations ranging from 1 to 100 pM with
50 nM ULight-PKC substrate and 10 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 10 pM PKC-␣
and 50 nM ULight-PKC substrate. 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 30 pM to 3 µM (final concentrations in
1% DMSO) were incubated with 10 pM PKC-␣, 50 nM ULight-PKC substrate and
10 µM ATP. Kinase reactions were terminated after 30 min by the addition of EDTA.
PKC-␣ enzyme at 10 pM was incubated with 50 nM ULight-PKC substrate and
10 µM ATP with or without 1 µM staurosporine (final concentrations in 1% DMSO).
Kinase reactions were terminated after 30 min by the addition of EDTA.
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©2007 PerkinElmer, Inc. All rights reserved. The PerkinElmer logo and design are registered trademarks of PerkinElmer, Inc. OptiPlate, TopSeal and ULight are trademarks and EnVision and
LANCE are registered trademarks of PerkinElmer, Inc. or its subsidiaries, in the United States and other countries. All other trademarks not owned by PerkinElmer, Inc. or its subsidiaries that are
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or typographical errors.
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