TECH NOTE U-TRF #14 INSR Kinase Assay

ULight-IRS-1 (Tyr983) Peptide and
Europium-anti-phospho-tyrosine Antibody (PT66)
Two LANCE Ultra companion products – two convenient sizes!
ULight™-IRS-1 (Tyr983) Peptide:
• TRF0120-M: 10 nmoles, 10,000* assay points
• AD0068: 50 µg, 7,500* assay points
*1 pmol/assay point
• AD0069: 1 mg, 150,000* assay points
PEPTIDE SEQUENCE:
*40 fmol/assay point
CKKSRGDYMTMQIG
RECOGNIZED MOTIF:
Synthetic peptide derived from residues 979-989 of mouse
Insulin receptor substrate 1 (IRS-1); phosphorylation site: Tyr983.
pTyr
VALIDATED FOR KINASES: INSR, EphB4, FGFR1, FLT1,
IGFR1, JAK1, JAK2, PDGFRa, RET, TIE2, TYK2
Mouse monoclonal antibody directed against phosphotyrosine.
POTENTIAL SUBSTRATE FOR KINASES:
Tyrosine kinases specific for the YMxM motif
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 an INSR Kinase Assay
Additional reagents
INSR active
Carna # 08-242
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
• TRF0120-D: 1 nmole, 1,000* assay points
Europium-anti-phospho-tyrosine Antibody
(PT66):
T E C H N I C A L
INSR Kinase Assay
L A N C E U LT R A
TECH NOTE U-TRF #14
Suggested procedure
• Dilute the INSR kinase, ATP, inhibitors and ULight-IRS-1 (Tyr983)
peptide in Kinase Buffer.
• Prepare a 4X Detection Mix by diluting the Eu-anti-phosphotyrosine antibody (PT66) to 8 nM in 1X LANCE Detection Buffer.
• Add to the wells of a white Optiplate-384:
– 5 µL of INSR enzyme
– 2.5 µL of inhibitor or Kinase Buffer
– 2.5 µL of ULight-IRS-1 (Tyr983) 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 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–tyrosine
Antibody (PT66) 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.
Experiment 1: Enzymatic Time Course
Experiment 2: ATP Titration
INSR enzyme was incubated at concentrations ranging from 2.5 to 20 nM with
100 nM ULight-IRS-1 (Tyr983) peptide and 1 mM ATP. Kinase reactions were
terminated after 0 to 240 min by the addition of EDTA.
Serial dilutions of ATP ranging from 10 nM to 1 mM were added to 10 nM INSR
and 100 nM ULight-IRS-1 (Tyr983) peptide. Kinase reactions were terminated
after 4 hours 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 1% DMSO) were incubated with 10 nM INSR, 100 nM ULight-IRS-1 (Tyr983) and
1 mM ATP. Kinase reactions were terminated after 4 hours by the addition of EDTA.
INSR enzyme at 10 nM was incubated with 100 nM ULight-IRS-1 (Tyr983) peptide
and 1 mM ATP with or without 10 µM staurosporine (final concentrations in 1%
DMSO). Kinase reactions were terminated after 4 hours by the addition of EDTA.
These results show that LANCE Ultra reagents can be used to monitor the activity of kinases such as INSR with very high Km for ATP.
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