IP Kinase Activity Assay Kit
Catalog # KNZ6031
OmniaTM
Agarose Bead IP
Kit for ERK1/2
BioSource International, Inc.
542 Flynn Road
Camarillo, California 93012 USA
Tel: 805-987-0086 • 800-242-0607
FAX: 805-987-3385
email: tech.support@biosource.com
BioSource Europe S.A.
Rue de l’Industrie, 8
B-1400 Nivelles, Belgium
Tel: +32 67 88 99 99
FAX: +32 67 88 99 96
email: tech.support@biosource.be
1
2
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 ............................................................
OmniaTM Agarose Bead IP Kinase Assay Kit for ERK1/2
Sample Data ................................................................................
References ...................................................................................
Patents, Trademarks, Limitations of Use.....................................
3
Rev. A1
12/14/06
PR377
4
5
8
9
9
11
12
12
12
14
15
17
20
20
INTRODUCTION
The mitogen-activated protein kinase (MAPK) signaling pathways
serve as pivotal transducers of diverse biologic functions including cell
growth, differentiation, proliferation, and apoptosis1. There are at least
three distinct MAPK signaling modules which mediate extracellular
signals into the nucleus to turn on the responsive genes in mammalian
cells, including extracellular mitogen-regulated kinase (ERK), c-Jun
NH2-terminal kinase (JNK, also called stress-activated protein kinase,
SAPK), and p38 kinase. The ERK signaling transduction pathway plays
an essential role in regulating many cellular processes including cell
differentiation and cell growth2.
ERK has two closely related isoforms of 44 kDa (ERK1) and 42 kDa
(ERK2), respectively. These kinases belong to a family of serine/
threonine kinases that are activated upon treatment of cells with a large
variety of stimuli including mitogens, hormones, growth factors,
cytokines, and bioactive peptides3. Cell stimulation induces the
activation of a signaling cascade, the downstream effects of which have
been linked to the regulation of cell growth and differentiation as well as
the cytoskeleton4. ERK1 and ERK2 are fully activated by
phosphorylation within a Thr-Glu-Tyr motif of the activation loop on
both a threonine and a tyrosine residue by MEKs (MAPK/ERK
kinases), thereby greatly elevating the activity of the ERK1/2 enzymes5.
Activated ERK phosphorylates numerous substrates in all cellular
compartments, including various membrane proteins, nuclear substrates,
and cytoskeletal proteins6.
This kit has been configured for research use only and is not
to be used in diagnostic procedures.
4
READ ENTIRE PROTOCOL BEFORE USE
PRINCIPLE OF THE METHOD
The OmniaTM Agarose Bead IP Kinase Assay Kit for ERK1/2 is
designed to measure the kinase activity of ERK1/2 from cell lysates.
This kit uses an ERK1/2 specific monoclonal antibody to capture the
target from the complex mixture of proteins in a crude cell lysate. The
phosphotransferase (kinase) activity of the captured ERK1/2 is
measured using a novel peptide substrate that contains the chelationenhanced fluorophore, 8-hydroxy-5-(N, N-dimethylsulfonamido)-2methylquinoline (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 using
standard solid-phase peptide chemistry8. Upon phosphorylation of the
peptide by ERK1/2, Mg++ is chelated to form a bridge between the Sox
moiety and the phosphate group that is added by ERK1/2 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.
5
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
6
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
peptide substrate (lower curve) and Sox 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 peptide
substrate (lower curve) and Sox phosphopeptide product (upper curve)
in the presence of 15 mM MgCl2, showing the characteristic increase in
fluorescence upon phosphorylation, as measured using a constant
excitation wavelength of 360 nm (for Graphs B and C, data for the
OmniaTM S/T Peptide 1 are shown as an example; fluorescence
increases with only OmniaTM substrates may vary). 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.
7
REAGENTS PROVIDED
Note: Store ERK1/2 Specific Monoclonal Antibody, ATP, DTT,
OmniaTM S/T Peptide 17 Solution, OmniaTM S/T Phosphopeptide 17
Solution and OmniaTM Cell Lysis Buffer at -20°C, Protein A & G
Agarose Beads at 2-8°C, 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 OmniaTM Agarose Bead IP Kinase Assay Kit for ERK1/2 is
designed to allow 40 assays (in 100 µL assay volume) to be performed
in a 96-well plate.
Description
Formula
Amount
OmniaTM Cell Lysis Buffer (1x)
Proprietary formulation developed to 30 mL
provide optimum enzyme activity
Wash Buffer Concentrate (10x)
Proprietary formulation developed to 15 mL
provide optimum enzyme activity
OmniaTM Kinase Reaction Buffer
Concentrate (10x)
Proprietary formulation developed to 10 mL
provide optimum enzyme activity
ERK1/2 Specific Monoclonal
Antibody Solution
Protein A & G Agarose Beads
100 µg/mL in Antibody Dilution
Buffer
Suspension containing 50% beads
slurry in PBS
500 µL
OmniaTM S/T Peptide 17 Solution
(50x)
OmniaTM S/T Phosphopeptide 17
Solution (50x)
Sox modified peptide substrate for
ERK1/2, 1 mM solution in water
Sox modified ERK1/2
phosphopeptide, 1 mM solution in
water (positive control)
100 mM ATP solution in water
100 mM DTT solution in water
200 µL
ATP Solution (100x)
DTT Solution (500x)
800 µL
20 µL
100 µL
200 µL
8
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.
9
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 2 (Cat. # P-2850, P-5726).
7. ERK1 enzyme (available from Invitrogen, Cat. # PV3311) can be
used for positive experimental controls and to compare the
ERK1/2 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 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).
10
PROCEDURAL NOTES
1.
2.
3.
4.
5.
6.
7.
8.
9.
11
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
ERK1/2 present in the cell lysate used in the study.
PROTOCOL AND RECOMMENDED ASSAY PROCEDURES
A.
Procedure for Cell Lysis Buffer Preparation
The OmniaTM 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 OmniaTM Cell Lysis Buffer should be thawed on ice.
Important: Add the protease inhibitors just before using. The stability
of protease inhibitor supplemented OmniaTM 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
ERK1/2 to determine ERK1/2 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 OmniaTM Cell Lysis Buffer on ice.
Set up and stimulate cells as desired.
12
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 OmniaTM Cell Lysis Buffer and
transfer the lysate to a 1.5 mL microcentrifuge tube. The volume of
OmniaTM Cell Lysis Buffer depends on the cell number and
expression level of ERK1/2. 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 ERK1/2 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.
13
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 Concentrate 10-fold with ultrapure water
(e.g., 5 mL of 10x Wash Buffer + 45 mL of ultrapure water).
OmniaTM Kinase Reaction Buffer (prepare 1x stock): Dilute an
appropriate amount of the 10x OmniaTM 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 OmniaTM Kinase
Reaction Buffer + 10 µL of 100 mM DTT solution + 4,490 µL
ultrapure water).
OmniaTM S/T Peptide 17 Solution (prepare 100 µM stock): Dilute
an appropriate amount of the provided peptide solution (1 mM)
10-fold with 1x OmniaTM Kinase Reaction Buffer (e.g., 10 µL of
1 mM peptide + 90 µL of 1x OmniaTM Kinase Reaction Buffer).
ATP Solution (prepare 5 mM stock): Dilute an appropriate amount
of 100 mM ATP solution 20-fold with 1x OmniaTM Kinase
Reaction Buffer (e.g., 10 µL of 100 mM ATP + 190 µL of 1x
OmniaTM Kinase Reaction Buffer).
Cell lysates: Dilute the lysate to 0.5 to 1 mg/mL total protein with
OmniaTM Cell Lysis Buffer. The amount of cell lysate protein used
in the assay varies depending on the quantity and activity of
ERK1/2 in the individual cell line.
14
6.
ERK1 kinase: Recombinant ERK1 enzyme can be used as a
positive control to quantify the activity of ERK1/2 in the cell
lysates. We recommend using the product from Invitrogen
(Cat. # PV3311). Dilute the ERK1 kinase to 4 ng/µL with 1x
OmniaTM Kinase Reaction Buffer and store on ice until use. We
recommend using 20 mU of ERK1 prepared in 10 µL of OmniaTM
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.
15
Add 10 µL of anti-ERK1/2 antibody solution to each tube
containing 50 - 400 µg of total cell lysate protein prepared in
500 µL OmniaTM 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.
Wash the beads again with 1 mL of 1x OmniaTM Kinase Reaction
Buffer.
8. 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 OmniaTM 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 S/T
Peptide 17 Solution and 20 µL of 5 mM ATP, each prepared in 1x
OmniaTM Kinase Reaction Buffer. The final concentration of
OmniaTM S/T Peptide 17 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.
7.
16
OMNIATM AGAROSE BEAD IP KINASE ASSAY KIT FOR
ERK1/2 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.
50000
40000
RFU
PDGF-Treated NIH3T3 Cell Lysate, 400 µg
Control-Treated NIH3T3 Cell Lysate, 400 µg
30000
20 µL Beads Only, No Ab. Control
20 µL Beads, 1 µg ERK1/2 Ab., No Lysate Control
20000
10000
0
60
120
180
240
300
Time (minutes)
Figure 2. Measurement of ERK1/2 Activity in Crude Lysates from
PDGF-Treated or Control NIH3T3 Cells. NIH3T3 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 serumfree medium to induce quiescence, followed by treatment with PDGF
A/B (50 ng/mL, 10 min) or control media. Cell lysates were prepared
and ERK1/2 activity was assayed as described in the Assay Procedure
section (page 15). The PDGF-treated sample showed a reaction rate of
1.55 RFU/sec, whereas the control treated sample had a markedly
reduced rate of 0.27 RFU/sec, resulting in a dramatic signal-to-noise
ratio of 5.7. Other assay control groups (bead only group or bead and
antibody only group) also had reaction rates less than 0.27 RFU/sec.
17
A.
60000
800 µg PDGF-Treated NIH 3T3 Cell Lysate
400 µg PDGF-Treated NIH 3T3 Cell Lysate
50000
200 µg PDGF-Treated NIH 3T3 Cell Lysate
RFU
40000
100 µg PDGF-Treated NIH 3T3 Cell Lysate
800 µg Control-Treated NIH 3T3 Cell Lysate
30000
400 µg Control-Treated NIH 3T3 Cell Lysate
200 µg Control-Treated NIH 3T3 Cell Lysate
20000
100 µg Control-Treated NIH 3T3 Cell Lysate
10000
20 µL Beads no Ab. Control
20 µL Beads + 1 µg ERK1/2 Ab. No Lysate
0
0
60
120
180
240
300
Time (minutes)
1.6
B.
Reaction Rate (RFU/Sec)
1.5
1.4
1.3
1.2
1.1
1
0.9
0.8
0.7
0
100
200
300
400
PDGF-Treated NIH 3T3 Cell Lysate (µg)
Figure 3. Comparison of ERK1/2 Activity Using Different Amounts
of Cell Lysate. A. PDGF-treated NIH3T3 lysates (800, 400, 200,
100 µg) were tested for ERK1/2 activity in comparison with the control
cell lysates. B. The reaction rates of the PDGF-treated samples are
directly proportional to the amounts of the protein in the samples from
50 to 400 µg, with a coefficient of correlation (R2) of 0.96.
18
50000
45000
40000
PDGF-Treated NIH3T3 Lysate - 1
PDGF-Treated NIH3T3 Lysate - 2
35000
PDGF-Treated NIH3T3 Lysate - 3
RFU
30000
PDGF-Treated NIH3T3 Lysate - 4
PDGF-Treated NIH3T3 Lysate - 5
25000
Control-Treated NIH3T3 Lysate - 1
20000
Control-Treated NIH3T3 Lysate - 2
15000
Control-Treated NIH3T3 Lysate - 3
Control-Treated NIH3T3 Lysate - 4
10000
Control-Treated NIH3T3 Lysate - 5
5000
0
0
50
100
150
200
250
300
Time (minutes)
Figure 4. Reproducibility of the OmniaTM Agarose Bead IP Kinase
Assay Kit for ERK1/2. Replicates (n = 5) of PDGF-treated NIH3T3
cell lysate samples (400 µg each) were tested in comparison with the
control cell lysates. The PDGF-treated group showed a reaction rate of
1.57 ± 0.06 RFU/sec (% CV = 1.27%), whereas the control group had a
rate of 0.33 ± 0.01 RFU/sec (% CV = 3.03%), illustrating the high
precision obtained with the OmniaTM platform.
19
REFERENCES
1.
2.
3.
4.
5.
6.
7.
8.
Seger, R. and Krebs, E.G. (1995) The MAPK signaling cascade.
FASEB J. 9:726-735.
Cavanaugh, J.E., et al. (2001) Differential regulation of mitogenactivated protein kinases ERK1/2 and ERK5 by neurotrophins,
neuronal activity, and cAMP in neurons. J. Neurosci. 21:434-443.
Chu, B., et al. (2005) Regulation of ERK1 gene expression by
coactivator proteins. Biochem. J. 392:589-599.
Kohno, M. and Pouyssegur, J. (2003) Pharmacological inhibitors
of the ERK signaling pathway: application as anticancer drugs.
Prog. Cell Cycle Res. 5:219-224.
Canagarajah B. J., et al. (1997) Activation mechanism of the MAP
kinase ERK2 by dual phosphorylation. Cell 90:859-869.
Chen, Z., et al. (2001) MAP kinases. Chem. Rev. 101:2449-2476.
Shults, M.D. and Imperiali, B. (2003) Versatile fluorescence
probes of protein kinase activity. J. Am. Chem. Soc.
125:14248-14249.
Shults, M.D., et al. (2005) A multiplexed homogeneous
fluorescence-based assay for protein kinase activity in cell lysates.
Nat. Methods 2:277-283.
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.
20
NOTES
21
22
23
OmniaTM IP Kinase Assay Summary
24
Rev. A1
12/14/06
PR377