IP Kinase Activity Assay Kit
Catalog # KNZ6041
Omnia®
Agarose Bead IP Kit for
MAPKAP-K2
www.invitrogen.com
Invitrogen Corporation
Carlsbad, California 92008
Tel: 800-955-6288
E-mail: techsupport@invitrogen.com
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TABLE OF CONTENTS
Introduction .................................................................................
Principle of the Method.................................................................
Reagents Provided ........................................................................
Safety Precautions .......................................................................
Supplies Required But Not Provided .............................................
Procedural Notes .........................................................................
Protocol and Recommended Assay Procedures...........................
A. Cell Lysis Buffer Preparation ........................................
B. Extraction of Proteins from Cells...................................
C. Assay Reagent Preparation ............................................
D. Assay Procedure ............................................................
Omnia® Agarose Bead IP Kinase Assay Kit for MAPKAP-K2
Sample Data ................................................................................
References ...................................................................................
Patents, Trademarks, Limitations of Use.....................................
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Rev. A1
10/12/07
PR416
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INTRODUCTION
MAP kinase-activated protein kinase 2 (MAPKAP-K2 or MK2) is a
stress-activated enzyme1,2. It is activated by MAPK family kinases, such
as p38 MAPK, or extracellular signal-regulated kinases 1 and 2
(ERK1/2)3. It transduces signals to target proteins that are not direct
substrates of the MAPKs, relaying phosphorylation-dependent signaling
within MAPK cascades to diverse cellular functions, such as
lipopolysaccharide-induced upregulation of cytokine mRNA stability,
cell-cycle regulation, cell migration and reorganization of the
cytoskeleton4,5. MAPKAP-K2 is implicated in inflammation and in
several disorders including heart failure and brain ischemic injury. In
mammalian cells, it is responsible for phosphorylation of small heat
shock protein, tyrosine hydroxylase, leukocyte specific protein 1,
5-lipoxigenase, SRF and E47. Its involvement in lipopolysaccharideinduced biosynthesis of several pro-inflammatory and inflammatory
cytokines is mediated through the phosphorylation of AU-rich elements
(AREs) -binding proteins6.
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PRINCIPLE OF THE METHOD
The Omnia® Agarose Bead IP Kinase Assay Kit for MAPKAP-K2 is
designed to measure the kinase activity of MAPKAP-K2 from cell
lysates. This kit uses an MAPKAP-K2 -specific antibody to capture the
target from the complex mixture of proteins in a crude cell lysate. The
phosphotransferase (kinase) activity of the captured MAPKAP-K2 is
measured using a novel peptide substrate that contains the chelationenhanced fluorophore, 8-hydroxy-5-(N, N-dimethylsulfonamido)-2methylquinoline (referred to as Sox7 ) in a real-time kinetic
measurement mode. Sox is an unnatural amino acid that can be prepared
as an Fmoc protected derivative, and has been incorporated into the
substrate peptide (Omnia® Ser/Thr Peptide 3) using standard
solid-phase peptide chemistry9. Upon phosphorylation of the peptide by
MAPKAP-K2, Mg++ is chelated to form a bridge between the Sox
moiety and the phosphate group that is added by MAPKAP-K2 to the
serine residue within the peptide, resulting in an instantaneous increase
in fluorescence when the kinase reaction mixture is excited at 360 nm
and the emission is measured at 485 nm7.
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A.
B.
C.
12000
Relative Fluorescence Units
Relative Fluorescence Units
12000
10000
8000
6000
4000
2000
0
290
310
330
350
370
Wavelength, nm
390
410
10000
8000
6000
4000
2000
0
410
460
510
560
610
Wavelength, nm
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Figure 1. A. Schematic view of Mg++ chelation by Sox and the
phosphate group on the modified serine, threonine or tyrosine residue in
the resulting phosphopeptide. B. Fluorescence excitation spectra of Sox
Akt1 peptide substrate (lower curve) and Sox Akt1 phosphopeptide
product (upper curve) in the presence of 15 mM MgCl2, as measured
using an emission wavelength of 485 nm. C. Fluorescence emission
spectra of Sox Akt1 peptide substrate (lower curve) and Sox Akt1
phosphopeptide product (upper curve) in the presence of 15 mM MgCl2,
showing the characteristic 10-fold increase in fluorescence upon
phosphorylation, as measured using a constant excitation wavelength of
360 nm. The non-phosphorylated version of the Sox-modified peptide
substrate has a very low affinity for Mg++ (KD = 100 - 300 mM). The
affinity for Mg++ increases dramatically upon phosphorylation
(KD = 4 - 20 mM). Therefore, upon phosphorylation, most of the
phosphopeptide exists in the Mg++-chelated, fluorescent state in the
presence of 15 mM MgCl2.
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REAGENTS PROVIDED
Note: Store MAPKAP-K2 Specific Antibody, ATP, DTT, Ser/Thr
Peptide 3, Ser/Thr Phosphopeptide 3 and Cell Lysis Buffer at -20°C,
Protein A & G Agarose Beads at 2-8°C, and Wash Buffer at room
temperature. We recommend that the vials provided be briefly
centrifuged prior to opening to bring the contents to the bottom.
The Omnia® Agarose Bead IP Kinase Assay Kit for MAPKAP-K2 is
designed to allow 40 assays (in 100 µL assay volume) to be performed
in a 96-well plate.
Description
Formula
Amount
Cell Lysis Buffer (1x)
Proprietary formulation developed to 30 mL
provide optimum enzyme activity
Wash Buffer (10x)
Proprietary formulation developed to 15 mL
provide optimum enzyme activity
Kinase Reaction Buffer (10x)
Proprietary formulation developed to 10 mL
provide optimum enzyme activity*
MAPKAP-K2 Specific Antibody
Solution
Protein A & G Agarose Beads
400 µg/mL in Antibody Dilution
Buffer
Suspension containing 50% beads
slurry in PBS
500 µL
800 µL
Sox modified peptide substrate for
200 µL
MAPKAP-K2, 1 mM solution in
water
Ser/Thr Phosphopeptide 3 (50x)
Sox modified MK2 phosphopeptide, 20 µL
1 mM solution in water (positive
control)
ATP Solution (100x)
100 mM ATP solution in water*
100 µL
DTT Solution (500x)
100 mM DTT solution in water*
200 µL
* These solutions contain 0.05% sodium azide as a preservative.
Ser/Thr Peptide 3 (50x)
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SAFETY PRECAUTIONS
This kit contains small quantities of sodium azide. Sodium azide reacts
with lead and copper plumbing to form explosive metal azides. Upon
disposal, flush drains with a large volume of water to prevent azide
accumulation. Avoid ingestion and contact with eyes, skin and mucous
membranes. In case of contact, rinse affected area with plenty of water.
Observe all federal, state and local regulations for disposal.
All biological materials should be handled as potentially hazardous.
Follow universal precautions as established by the Centers for Disease
Control and Prevention and by the Occupational Safety and Health
Administration when handling and disposing of potentially hazardous
materials.
SUPPLIES REQUIRED BUT NOT PROVIDED
1.
2.
3.
4.
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Fluorescence microplate reader capable of excitation wavelength at
360 nm, emission wavelength of 485 nm, and measurements in a
kinetic manner (e.g., ability to take readings every 30 seconds over
a 5 hour time period). This kit was developed using a SpectraMax
M5® microplate reader from Molecular Devices, although other
comparable instruments are acceptable.
Microtiter plate for reading fluorescent signals. We recommend
NBSTM 96-well Microplate (Cat. # 3992) from Corning Inc., which
is a non-protein binding, white solid plastic, half well flat bottom
plate.
Calibrated adjustable precision pipettes with disposable plastic tips.
A manifold multi-channel pipette is desirable for processing a
large number of assays.
Ultrapure (18MΩ) deionized H2O.
5.
Plastic tubes with low protein binding for diluting and aliquoting
assay components.
6. Protease and phosphatase inhibitors. We recommend Sigma
Protease Inhibitor Cocktail (Cat. # P-8340) and Sigma Phosphatase
Inhibitor Cocktail (Cat. # P-2850, P-5726).
7. MAPKAP-K2 enzyme (available from Invitrogen, Cat. # PV3317)
can be used for positive experimental controls and to compare the
MAPKAP-K2 activity from cell lysates.
8. Rocking platform, shaker or rotator with a rate of 5 to 100
oscillations per minute.
9. Ultrasonic homogenizer or a 19 gauge needle and a 5 mL syringe
for breaking up the cells.
10. Microcentrifuge with a spin speed up to 14,000 rpm (18,000 x g).
11. Quantitative protein assay kits. We recommend the Quant-iTTM
Assay Kit from Invitrogen (Cat. # Q33210).
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PROCEDURAL NOTES
1.
2.
3.
4.
5.
6.
7.
8.
9.
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When not in use, certain kit components need to be stored at
-20°C. Please follow the recommendations for storage condition as
required. All frozen reagents should be thawed on ice before use.
Samples should be frozen if not analyzed shortly after collection.
Avoid multiple freeze-thaw cycles of frozen samples. Thaw
completely and mix well prior to analysis.
If particulate matter is present, centrifuge or filter prior to analysis.
All standards, controls, and samples should be run in duplicate.
When pipetting reagents, maintain a consistent order of addition
from well-to-well. This ensures equal incubation times for all
wells.
Cover or cap all reagents when not in use.
Do not mix or interchange different reagent lots from various kit
lots.
Do not use reagents after the kit expiration date.
The starting time for reading the fluorescence signal in a plate
reader can be varied from 0 to 1 hour, depending on the quantity of
MAPKAP-K2 present in the cell lysate used in the study.
PROTOCOL AND RECOMMENDED ASSAY PROCEDURES
A.
Procedure for Cell Lysis Buffer Preparation
The Cell Lysis Buffer provided in this kit (also available separately,
Cat. # CE001A) needs to be supplemented with phosphatase inhibitors
(such as Phosphatase Inhibitor Cocktail, Sigma Cat. # P-2850, P-5726)
and protease inhibitors (such as PMSF or AEBSF, 1 mM; Protease
Inhibitor Cocktail, Sigma Cat. # P-8340) according to manufacturer’s
recommendations.
This buffer is stable for 2-3 weeks at 4°C or for up to 18 months at
-20°C (without protease or phosphatase inhibitors added). When stored
frozen, the Cell Lysis Buffer should be thawed on ice.
Important: Add the protease inhibitors just before using. The stability
of protease inhibitor supplemented Cell Lysis Buffer is 24 hours at 4oC.
PMSF is very unstable and must be re-added just prior to use, even if
added previously.
B.
Procedure for Extraction of Proteins from Cells
When using the OmniaTM Agarose Bead IP Kinase Assay Kit for
MAPKAP-K2 to determine MAPKAP-K2 activity in cell lysates, we
recommend the following procedure for sample preparation. This
protocol has been successfully applied to several cell lines of human and
mouse origin. Researchers should optimize the cell extraction
procedures for their own applications.
1.
2.
Thaw Cell Lysis Buffer on ice.
Set up and stimulate cells as desired.
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3.
Collect cells in cold PBS by centrifugation (for non-adherent cells)
or scraping from culture plates (for adherent cells).
4. Centrifuge the cells at 1,500 rpm for 5 minutes at 4°C.
5. Aspirate the PBS.
6. Resuspend the cell pellet in 1x Cell Lysis Buffer and transfer the
lysate to a 1.5 mL microcentrifuge tube. The volume of Cell Lysis
Buffer depends on the cell number and expression level of
MAPKAP-K2. The optimal protein concentration of lysate should
be in the range of 5 to 20 mg/mL or approximately 20 – 80 x 106
cells/mL. Add an appropriate amount of protease and phosphatase
inhibitors (typically provided as a 100x stock solution) before
using. Under these conditions, using 10 µL (50-200 µg) of the
clarified cell extract should be sufficient for measurement of
MAPKAP-K2 activity.
7. Lyse the cells at 4°C for 30 minutes on a rotator. Whole cell
extract then can be briefly sonicated or put through a syringe and
needle if desired.
8. Centrifuge at 13,000 rpm for 30 minutes at 4°C.
9. Transfer the clarified cell extracts to clean microcentrifuge tubes.
Determine the total protein concentration using an accepted
procedure, such as the Quant-iTTM Assay Kit from Invitrogen
(Cat. # Q33210).
10. The clarified cell extract should be stored at -80°C until ready for
analysis. Avoid repeated freeze-thaw cycles. In preparation for
performing the assay, allow the samples to thaw on ice. Mix well
prior to analysis.
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C.
Procedure for Assay Reagent Preparation
Prior to setting up the individual reactions, the following solutions must
be prepared:
1.
2.
3.
4.
5.
Wash Buffer (prepare 1x stock): Dilute an appropriate amount of
the 10x Wash Buffer 10-fold with ultrapure water (e.g., 5 mL of
10x Wash Buffer + 45 mL of ultrapure water).
Kinase Reaction Buffer (prepare 1x stock): Dilute an appropriate
amount of the 10x Kinase Reaction Buffer 10-fold with ultrapure
water and add DTT (provided) to a final concentration of 0.2 mM
(e.g., 500 µL of 10x Kinase Reaction Buffer + 10 µL of 100 mM
DTT solution + 4,490 µL ultrapure water).
Ser/Thr Peptide 3 (prepare 100 µM stock): Dilute an appropriate
amount of the provided peptide solution (1 mM) 10-fold with 1x
Kinase Reaction Buffer (e.g., 10 µL of 1 mM peptide + 90 µL of
1x Kinase Reaction Buffer).
ATP Solution (prepare 5 mM stock): Dilute an appropriate amount
of 100 mM ATP solution 20-fold with 1x Kinase Reaction Buffer
(e.g., 10 µL of 100 mM ATP + 190 µL of 1x Kinase Reaction
Buffer).
Cell lysates: Dilute the lysate to 0.5 to 1 mg/mL total protein with
Cell Lysis Buffer. The amount of cell lysate protein used in the
assay varies depending on the quantity and activity of
MAPKAP-K2 in the individual cell line.
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6.
MAPKAP-K2 kinase: Recombinant MAPKAP-K2 enzyme can be
used as a positive control to quantify the activity of MAPKAP-K2
in the cell lysates. We recommend using the product from
Invitrogen (Cat. # PV3317). Dilute the MAPKAP-K2 kinase to
4 ng/µL with 1x Kinase Reaction Buffer and store on ice until use.
We recommend using 10 to 30 ng of MAPKAP-K2 prepared in
10 µL of Kinase Reaction Buffer for each reaction.
D.
Assay Procedure
Be sure to read the Procedural Notes section before carrying out the
assay.
Thaw frozen reagents on ice. Allow all reagents to reach room
temperature before use. Gently mix all liquid reagents prior to use.
1.
2.
3.
4.
5.
6.
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Add 10 µL of anti-MAPKAP-K2 antibody solution to each tube
containing 50 - 400 µg of total cell lysate protein prepared in
500 µL Cell Lysis Buffer, and incubate overnight at 4ºC on a
rocking platform.
Add 20 µL Protein A & G Agarose bead suspension (50% slurry in
PBS) to each of the tubes, and incubate for a minimum of 2 hours
at 4ºC on a rocking platform.
Collect the beads by centrifugation at 10,000 rpm (12,000 x g) for
10 seconds in a microcentrifuge.
Remove supernatant carefully by decanting or aspiration; add
1 mL of cold Wash Buffer to resuspend the beads.
Repeat Steps 3 and 4 one more time for a total of 2 times.
Remove the last trace of the Wash Buffer from the tubes using a
pipette tip.
7.
8.
Wash the beads again with 1 mL of 1x Kinase Reaction Buffer.
Collect beads by centrifugation at 10,000 rpm (12,000 x g) for
10 seconds in a microcentrifuge. Remove the supernatant from the
tubes using a pipette tip.
9. Resuspend the beads with 50 µL of 1x Kinase Reaction Buffer and
transfer the bead suspension to a well of an opaque 96-well plate
(such as Corning® Nonbinding Surface Microplates, Cat. # 3992).
10. To each of the sample wells, add 20 µL of 100 µM OmniaTM Ser/
Thr Peptide 3 Solution and 20 µL of 5 mM ATP, each prepared in
1x Kinase Reaction Buffer. The final concentration of Ser/Thr
Peptide 3 is 20 µM, and the final concentration of ATP is 1 mM.
The final reaction volume is 100 µL.
11. Transfer the plate to a fluorescence plate reader (such as
SpectraMax M5® by Molecular Devices, or a comparable
instrument). Read the fluorescence values of each well every
30 seconds at an excitation wavelength of 360 nm and an emission
wavelength of 485 nm for up to 5 hours at 30ºC in a kinetic mode.
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OMNIA® AGAROSE BEAD IP KINASE ASSAY KIT FOR
MAPKAP-K2 SAMPLE DATA
Note: All fluorescence intensity data are represented by relative fluorescence
units (RFU). These values are highly instrument and assay dependent, and should
not be considered to represent values that are universally applicable to all users
on all fluorescence detection instruments.
70000
60000
200 µg Anisomycin Treated RAW Lysate
50000
200 µg Control Lysate
RFU
40000
20 µL Beads Only, No MK2 Ab. Control
30000
20 µL Beads, 4 µg MK2 Ab.
No Lysate Control
20000
10000
0
0
30
60
90 120 150 180 210 240 270 300
Time (minutes)
Figure 2. Measurement of MAPKAP-K2 Activity in Crude Lysates
from Anisomycin-Treated or Control RAW Cells. RAW 264.7 cells were
seeded in 100 mm dishes and grown in DMEM plus 10% fetal bovine
serum until 90% confluent. The cells were then incubated overnight in
serum-free medium to induce quiescence, followed by treatment with
Anisomycin (10 µg/mL, 10 min) or control media. Cell lysates were
prepared and MAPKAP-K2 activity was assayed as described in the Assay
Procedure section (page 15). The Anisomycin-treated sample showed a
reaction rate of 2.62 RFU/sec, whereas the control treated sample had a
markedly reduced rate of 0.34 RFU/sec. Other assay control groups (bead
only group or bead and antibody only group) also had reaction rates less
than 0.34 RFU/sec.
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A.
70000
200 µg Anisomycin Treated Lysate
RFU
60000
100 µg Anisomycin Treated Lysate
50000
50 µg Anisomycin Treated Lysate
40000
25 µg Anisomycin Treated Lysate
200 µg Control Lysate
30000
100 µg Control Lysate
20000
50 µg Control Lysate
10000
25 µg Control Lysate
20 µL Beads, No MK2 Ab. Control
0
0
60
120
180
240
300
Time (minutes)
4.00
2
Reaction Rate (RFU/Sec.)
B.
R = 0.9686
3.50
3.00
2.50
2.00
1.50
0
50
100
150
200
250
Anisomycin-Treated RAW Cell Lysate (µg)
Figure 3. Comparison of MAPKAP-K2 Activity Using Different
Amounts of Cell Lysate. Anisomycin-treated RAW cell lysates were
tested for MAPKAP-K2 activity in comparison with untreated, control
cell lysates. The reaction rates of the Anisomycin-treated samples are
directly proportional to the amount of total protein in the samples from
25 to 200 µg, with a coefficient of correlation (R2) of 0.97.
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RFU
80000
Anisomycin-Treated RAW Lysate - 1
70000
Anisomycin-Treated RAW Lysate - 2
60000
Anisomycin-Treated RAW Lysate - 3
50000
Anisomycin-Treated RAW Lysate - 4
40000
Anisomycin-Treated RAW Lysate - 5
Control RAW Lysate - 1
30000
Control RAW Lysate - 2
20000
Control RAW Lysate - 3
10000
Control RAW Lysate - 4
0
0
60
120
180
240
300
Control RAW Lysate - 5
Time (minutes)
Figure 4. Reproducibility of the Omnia® Agarose Bead IP Kinase
Assay Kit for MAPKAP-K2. Replicates (n = 5) of Anisomycin
treated RAW cell lysate samples (200 µg each) were tested in
comparison with the control cell lysates. The Anisomycin-treated group
showed a reaction rate of 3.64 ± 0.16 RFU/sec (% CV = 5.4%), whereas
the control group had a rate of 0.88 ± 0.04 RFU/sec (% CV = 5.01%),
illustrating the high precision obtained with the OmniaTM platform.
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A.
80000
70000
Beads + MAPKAP-K2 Enzyme
60000
Beads + MAPKAP-K3 Enzyme
RFU
50000
40000
Beads + MAPKAP-K5 Enzyme
30000
Beads Only
20000
MK2tide
10000
0
0
20
40
60
80
100 120
Time (minutes)
B.
70000
60000
MAPKAP-K2 Enzyme
50000
MAPKAP-K3 Enzyme
RFU
40000
MAPKAP-K5 Enzyme
30000
20000
Beads Only
10000
MK2tide
0
0
30
60
90
120
Time (minutes)
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Figure 5. Selectivity of the Omnia® Agarose Bead IP Kinase Assay
Kit for MAPKAP-K2. A. Full length recombinant MAPKAP-K2,
MAPKAP-K3 and MAPKAP-K5 (80 mU each; Invitrogen Cat. #
PV3317, PV3299 and PV3301 respectively), were first incubated with
the beads and then their kinase activity was measured using the
protocol described in this booklet. Only MAPKAP-K2 showed activity
in the assay, illustrating the selectivity conferred by MAPKAP-K2
antibody used to capture MAPKAP-K2 from the cell lysate. B. Full
length recombinant MAPKAP-K2, MAPKAP-K3and MAPKAP-K5
(20 mU each) were tested for their kinase activity using a direct kinase
assay (without capture on the beads). This was a control to show that
all three enzymes exhibited the same level of activity in the assay.
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REFERENCES
1.
2.
3.
4.
5.
6.
7.
8.
9.
Gaestel, M. (2006) MAPKAPkinases — MKs — two’s
company, three’s a crowd. Nature Reviews Mol. Cell Biol.
7:120-130.
Roux, P.P. and Blenis, J. (2004) ERK and p38 MAPKActivated Protein Kinases: a Family of Protein Kinases with
Diverse Biological Functions. Microbiol. Mol. Biol. Rev.
68:320-344.
Freshney, N.W. et al. (1994) Interleukin-1 activates a novel
protein kinase cascade that results in the phosphorylation of
Hsp27. Cell 78:1039-1049.
Bulavin, D.V. et al. (2001) Initiation of a G2/M checkpoint alter
ultraviolet radiation requires p38 kinase. Nature 411:102-107.
Saklatvala, J. (2004) The p38 MAP kinase pathway as a
therapeutic target in inflammatory disease. Curr. Opin.
Pharmacol. 4:372-377.
Neininger, A. et al. (2002) MK2 targets AU-rich elements and
regulates biosynthesis of tumor necrosis factor and interleukin-6
independently at different post-transcriptional levels. J. Biol.
Chem. 277:3065-3068.
Shults, M.D. and Imperiali, B. (2003) Versatile fluorescence
probes of protein kinase activity. J. Am. Chem. Soc. 125
(47):14248-14249.
Obata, T. et al. (2000) Peptide and protein library screening
defines optimal substrate motifs for Akt/PKB. J. Biol. Chem.
275:36108-36115.
Shults, M.D. et al. (2005) A multiplexed homogeneous
fluorescence-based assay for protein kinase activity in cell lysates.
Nat. Methods 2:277-283.
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PATENTS, TRADEMARKS, LIMITATIONS OF USE
These products are sold under an exclusive license from the
Massachusetts Institute of Technology and are covered by patents
10/681,427 and 10/682,427.
Important Licensing Information - These products may be covered
by one or more Limited Use Label Licenses (see the Invitrogen
Catalog or our website, www.invitrogen.com). By use of these
products you accept the terms and conditions of all applicable Limited
Use Label Licenses. Unless otherwise indicated, these products are for
research use only and are not intended for human or animal diagnostic,
therapeutic or commercial use.
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Omnia® IP Kinase Assay Summary
24
Rev. A1
10/12/07
PR416