Tech Note U-TRF #13 P70 S6K Kinase Assay

ULight-mTOR (Ser2448) Peptide and
Europium-anti-phospho-mTOR (Ser2448) Antibody
T E C H N I C A L
p70 S6K Kinase Assay
L A N C E U LT R A
TECH NOTE U-TRF #13
Two LANCE Ultra companion products – two convenient sizes!
• TRF0119-D: 0.5 nmole, 1,000 assay points*
Europium-anti-phospho-mTOR
(Ser2448) Antibody:
• TRF0119-M: 5 nmoles, 10,000 assay points*
• TRF0209-D: 10 µg, 1,562 assay points*
*0.5 pmol/assay point
• TRF0209-M: 100 µg, 15,625 assay points*
PEPTIDE SEQUENCE:
*40 fmol/assay point
CAGAGRSRTRTDSYSAGQSV
RECOGNIZED MOTIF:
Synthetic peptide containing the residues surrounding Ser2448
of human mTOR; phosphorylation site: Ser2448.
RSRTRTDpSYSAGQSV
VALIDATED FOR KINASES: p70 S6K, MST1, IRAK4, JNK1,
JNK2, GSK-3B, MLK3
Europium-labeled rabbit monoclonal antibody recognizing
phospho-Ser2448 in human mTOR.
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 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 p70 S6K Kinase Assay
Additional reagents
p70 S6 Kinase, active
Upstate # 14-486
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™-mTOR (Ser2448) Peptide:
Suggested procedure
• Dilute the p70 S6K enzyme, ATP, inhibitors and ULight-mTOR
peptide in Kinase Buffer.
• Prepare a 4X Detection Mix by diluting the Eu-anti-phosphomTOR antibody to 8 nM in 1X LANCE Detection Buffer.
• Add to the wells of a white Optiplate-384:
– 5 µL of p70 S6K enzyme
– 2.5 µL of inhibitor or Kinase Buffer
– 2.5 µL of ULight-mTOR peptide/ATP mix
(for ATP titration, ATP dilutions are added
separately 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.
• Add 5 µL of 4X Detection Mix (Eu-anti-phospho-mTOR
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 Solution/Detection Mix to minimize
the number of liquid handling steps.
• Cover the plate with TopSeal-A and incubate at room
temperature (RT).
Experiment 1: Enzymatic Time Course
Experiment 2: ATP Titration
p70 S6K enzyme was incubated at concentrations ranging from 0.5 to 8 nM
with 50 nM ULight-mTOR 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 incubated with 2 nM
p70 S6K enzyme and 50 nM of ULight-mTOR peptide. Kinase reactions were
terminated after 45 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 30 µM (final concentrations
in 2% DMSO) were incubated with 2 nM p70 S6K enzyme, 50 nM ULight-mTOR
peptide and 3 µM ATP. Kinase reactions were terminated after 45 min by the
addition of EDTA.
p70 S6K enzyme at 2 nM was incubated with 50 nM ULight-mTOR peptide and
3 µM ATP with or without 1 µM staurosporine (final concentrations in 2% DMSO).
Kinase reactions were terminated after 45 min by the addition of EDTA.
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