IP Kinase Activity Assay Kit
Catalog # KNZ7051
Omnia®
Plate IP Kit
for Syk
www.invitrogen.com
Invitrogen Corporation
Carlsbad, California 92008
Tel: 800-955-6288
E-mail: techsupport@invitrogen.com
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 ............................................................
Omnia® Plate-Based IP Kinase Assay Kit for Syk
Sample Data ................................................................................
References ...................................................................................
Patents, Trademarks, Limitations of Use.....................................
3
Rev. A1
10/12/07
PR418
4
5
8
9
9
11
12
12
12
14
16
17
21
22
INTRODUCTION
Spleen tyrosine kinase (Syk) is a critical component of immunoreceptor
signaling following activation of B cell receptor or Fc receptors in
B cells, mast cells, macrophages and neutrophils1,2. It couples
immunoreceptors to transduction pathways that regulate phagocytosis,
cell differentiation, proliferation, adhesion, and motility. It also controls
calcium mobilization, phospholipase C function, Akt survival pathway
in B cells and integrin signaling in hematopoietic cells via both
phosphorylation-dependent and -independent processes3,4. Besides
blood cells, Syk is widely expressed in other cell types, such as
epithelial cells, hepatocytes, fibroblasts, neuronal cells, and vascular
endothelial cell lines, where it plays a variety of roles that are not yet
completely understood.
Syk is activated upon binding to immunoreceptor tyrosine-based
activating motif (ITAM)5. It contains two Src Homology 2 domains
located on the amino-terminal end of the protein. There are multiple
tyrosine residues in Syk that are auto-phosphorylated after its activation.
Phosphorylation at these tyrosine residues provides interaction sites for
the SH2 domain and regulates enzymatic activity in other molecules in
the downstream signaling pathway6.
4
PRINCIPLE OF THE METHOD
The Omnia® Plate-Based IP Kinase Assay Kit for Syk is designed to
measure the kinase activity of Syk in cell lysates. This kit uses a
Syk-specific antibody to capture the target and separate it from
the complex mixture of proteins in a crude cell lysate. The
phosphotransferase (kinase) activity of the captured Syk is measured by
using a novel peptide substrate which contains the chelation-enhanced
fluorophore, 8-hydroxy-5-(N,N-dimethyl sulfon-amido)-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 Syk
substrate peptide (Omnia® Tyr Peptide 7) using standard solid-phase
peptide chemistry8. Upon phosphorylation of the peptide by Syk, Mg++
is chelated to form a bridge between the Sox moiety and the phosphate
group that is added by Syk to the tyrosine 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 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) and Sox Akt1 phosphopeptide product (upper) 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.
7
REAGENTS PROVIDED
Note: Store ATP, DTT, Tyr Peptide 7, Tyr Phosphopeptide 7 and Cell
Lysis Buffer at -80°C, all other reagents at 2 to 8°C. We recommend
that the vials provided be briefly centrifuged prior to opening to bring
the contents to the bottom.
The Omnia® Plate-Based IP Kinase Assay Kit for Syk is designed to
allow 96 assays (in 100 µL assay volume) to be performed in a 96-well
plate.
Description
Cell Lysis Buffer (1x)
Formula
Amount
Proprietary formulation developed to 30 mL
provide optimum enzyme activity
Wash Buffer (10x)
Proprietary formulation developed to
provide optimum enzyme activity
15 mL
Tyr Kinase Reaction Buffer (10x)
Proprietary formulation developed to
provide optimum enzyme activity*
10 mL
Anti Syk Antibody-Coated Wells
12 x 8 well strips
Tyr Peptide 7 (50x)
Sox modified peptide substrate for
Syk, 1 mM solution in water
Tyr Phosphopeptide 7 (50x)
Sox modified Syk phosphopeptide,
1 mM solution in water (positive
control)
ATP Solution (100x)
100 mM ATP solution in water*
DTT Solution (500x)
100 mM DTT solution in water*
Adhesive Plate Cover Strips
Clear plastic sealing tape
* These solutions contain 0.05% sodium azide as a preservative.
96 wells
200 µL
20 µL
100 µL
200 µL
3
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.
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.
Plastic tubes with low protein binding for diluting and aliquoting
assay components.
5.
Protease and phosphatase inhibitors. We recommend Sigma
Protease Inhibitor Cocktail (Cat. # P-8340) and Sigma Phosphatase
Inhibitor Cocktails (Cat. # P-2850, P-5726).
6. Recombinant Syk enzyme (available from Invitrogen, Cat. #
PV3857) can be used for positive experimental controls and to
compare the Syk activity from cell lysates.
7. Rocking platform, shaker or rotator with a rate of 5 to 100
oscillations per minute.
8. Ultrasonic homogenizer or 19 gauge needle and 5 mL syringes for
breaking up the cells.
9. Microcentrifuge with a spin speed up to 14,000 rpm (18,000 x g).
10. Quantitative protein assay kits. We recommend the Quant-iTTM
Assay Kit from Invitrogen (Cat. # Q33210).
10
PROCEDURAL NOTES
1.
When not in use, certain kit components need to be stored at
-80°C. Please follow the recommendations for storage condition as
required. All frozen reagents should be thawed on ice before use.
2. Microtiter plate strips should be allowed to equilibrate to room
temperature before opening the foil bags. Once the desired number
of strips is removed, immediately reseal the bag and store at 2 to
8°C to maintain plate integrity.
3. 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.
4. If particulate matter is present, centrifuge or filter prior to analysis.
5. All standards, controls, and samples should be run in duplicate.
6. When pipetting reagents, maintain a consistent order of addition
from well-to-well. This ensures equal incubation times for all
wells.
7. Cover or cap all reagents when not in use.
8. Do not mix or interchange different reagent lots from various kit
lots.
9. Do not use reagents after the kit expiration date.
10. 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
Syk present in the cell lysate used in the study.
11. All residual wash liquid must be drained from the wells by
efficient aspiration or by decantation followed by tapping the plate
firmly on absorbent paper. Never insert absorbent paper directly
into the wells.
11
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 Cocktails, 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 to 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 Omnia® Plate-Based IP Kinase Assay Kit for Syk to
determine Syk 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.
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 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 Syk.
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 inhibitor
(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 Syk 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).
Tyr Kinase Reaction Buffer (prepare 1x stock): Dilute an
appropriate amount of the 10x Tyr 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 Tyr Kinase Reaction
Buffer + 10 µL of 100 mM DTT solution + 4,490 µL ultrapure
water).
Tyr Peptide 7 (prepare 100 µM stock): Dilute an appropriate
amount of the provided peptide solution (1 mM) 10-fold with 1x
Tyr Kinase Reaction Buffer (e.g., 10 µL of 1 mM peptide + 90 µL
of 1x Tyr Kinase Reaction Buffer).
ATP Solution (prepare 2 mM stock): Dilute an appropriate amount
of 100 mM ATP solution 50-fold with 1x Tyr Kinase Reaction
Buffer (e.g., 10 µL of 100 mM ATP + 490 µL of 1x Tyr 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 Syk in the
individual cell line.
14
6.
15
Syk kinase: Recombinant Syk enzyme can be used as a positive
control to quantify the activity of Syk in the cell lysates. We
recommend using the product from Invitrogen (Cat. # PV3857).
Dilute the Syk kinase to 4 ng/µL with 1x Tyr Kinase Reaction
Buffer and store on ice until use. We recommend using 10 to 50 ng
of Syk prepared in 10 µL of Tyr 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.
Add 100 µL of cell lysate containing 25 - 100 µg of total protein
prepared in Cell Lysis Buffer to each sample well in the Syk
antibody coated plate strips.
Seal the plate with Adhesive Plate Cover Strips and incubate at
room temperature for 2 hours.
Remove and discard the cell lysate from wells and wash plate 4
times with 200 µL of Wash Buffer. Aspirate or decant Wash
Buffer, and remove any residual liquid between washes by tapping
the plate firmly on absorbent paper.
Add 30 µL of Tyr Kinase Reaction Buffer, 20 µL of Tyr Peptide 7
(100 µM), and 50 µL of ATP (2 mM). The final concentration of
Tyr Peptide 7 in the reaction mixture is 20 µM and the final
concentration of ATP is 1 mM. The final volume of the reaction is
100 µL. Incubate at 30ºC for 1 hour.
Transfer the plate to a fluorescence plate reader (such as
SpectraMax M5® by Molecular Devices or 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.
16
OMNIA® PLATE-BASED IP KINASE ASSAY KIT FOR Syk
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.
5000
4000
3000
RFU
Anti-IgM Ab. Treated RAMOS Lysate, 100 µg
Control RAMOS Lysate, 100 µg
2000
Peptide only, No Lysate
1000
0
0
30
60
90
120
Time (minutes)
Figure 2. Measurement of Syk Activity in Crude Lysates from Anti-IgM
Antibody Treated or Control RAMOS Cells. RAMOS cells were seeded in
100 mm dishes and grown in RPMI 1640 medium with 2 mM L-glutamine
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 rabbit anti-human IgM antibody (Invitrogen, Cat. No. 62-7500; 20 µg/mL,
10 min) or control media. Cell lysates were prepared and Syk activity was
assayed as described in the Assay Procedure section (Page 16). The anti-IgM
antibody treated sample showed a reaction rate of 0.41 RFU/sec, whereas the
control treated sample had a markedly reduced rate of less than 0.01 RFU/sec.
The assay control group (peptide only) also had reaction rates of less than
0.01 RFU/sec.
17
A.
5000
Anti-IgM Ab. Treated RAMOS Lysate, 100 µg
Anti-IgM Ab. Treated RAMOS Lysate, 50 µg
4000
Anti-IgM Ab. Treated RAMOS Lysate, 25 µg
3000
RFU
Anti-IgM Ab. Treated RAMOS Lysate, 12.5 µg
Control RAMOS Lysate, 100 µg
2000
Control RAMOS Lysate, 50 µg
Control RAMOS Lysate, 25 µg
1000
Control RAMOS Lysate, 12.5 µg
0
Peptide Only, No Lysate Control
0
30
60
90
120
Time (minutes)
3000
B.
2500
RFU
2000
1500
1000
500
0
0
20
40
60
80
100
120
Lysate (µg)
Figure 3. Comparison of the Syk Activity Using Different Amounts of Cell
Lysate. A. Rabbit anti human IgM antibody-treated RAMOS cell lysates were
tested to compare their Syk activity in increasing amounts of total cell lysate
protein (0, 12.5, 25, 50, 100 µg) as described in the Assay Procedure Section. B.
Using 120 minutes as an arbitrary time point, the graph shows that the
fluorescence signals produced by these samples are directly proportional to the
amount of the protein (from 0 to 100 µg) in the sample, with a coefficient of
correlation (R2) of 0.94.
18
5000
Anti-IgM Ab. Treated RAMOS Lysate - 1
Anti-IgM Ab. Treated RAMOS Lysate - 2
4000
Anti-IgM Ab. Treated RAMOS Lysate - 3
Anti-IgM Ab. Treated RAMOS Lysate - 4
RFU
3000
Anti-IgM Ab. Treated RAMOS Lysate - 5
Control RAMOS Lysate - 1
2000
Control RAMOS Lysate - 2
Control RAMOS Lysate - 3
1000
Control RAMOS Lysate - 4
Control RAMOS Lysate - 5
0
0
30
60
90
120
Time (minutes)
Figure 4. Reproducibility of the Omnia® Plate-Based IP Kinase
Assay Kit for Syk. Replicates (n = 5) of rabbit anti-human IgM
antibody treated RAMOS cell lysate or untreated lysate control samples
(100 µg each) were tested for their Syk kinase activity as described in
the Assay Procedure Section. The anti-IgM antibody treated group
showed reaction rates of 0.47 ± 0.02 RFU/sec (% CV = 4.3%), whereas
the control group had reaction rates of less than 0.01 RFU/sec,
illustrating the high precision obtained with the Omnia® platform.
19
5000
Anti-IgM Ab. Treated RAMOS Lysate (100
µg)
3000
Non-specific IgG Depleted anti-IgM Ab.
Treated RAMOS Lysate (100 µg)
2000
Syk- Specific Ab. Depleted anti-IgM Ab.
Treated RAMOS Lysate Lysate (100 µg)
1000
Peptide Only Control, No Lysate
RFU
4000
0
0
30
60
90
120
Time (minutes)
Figure 5. Specificity of the Omnia® Plate-Based IP Kinase Assay
Kit for Syk. Rabbit anti-human IgM antibody treated RAMOS cell
lysate (100 µg) were incubated with non-specific mouse IgG antibody
(0.5 µg, LabVision), or Syk specific antibody (0.5 µg, LabVision) for
16 hours at 4°C. Agarose beads (20 µL, 50% slurry) were then added to
the incubation mixtures which were then agitated on a rocking platform
at 4°C for 2 hours. The incubation mixtures were then centrifuged to
remove Syk protein as an immune complex from the lysate. The
immunodepleted lysates were then tested for their Syk kinase
activity using Omnia® kinase assay as described Page 16. The kinase
activity was significantly reduced in IgM treated RAMOS cell lysate
that has been depleted with Syk specific antibody (reaction rate of
0.03 RFU/sec), in comparison with the non-specific mouse IgG treated
or non-treated RAMOS cell lysate (reaction rate of 0.35, 0.36 RFU/sec,
respectively).
20
REFERENCES
1.
2.
3.
4.
5.
6.
7.
8.
21
Berton, G. et al. (2005) Src and Syk kinases: key regulators of
phagocytic cell activation. Trends Immunol. 4:208-214.
Navara, C.S. (2004) The spleen tyrosine kinase (Syk) in human
disease, implications for design of tyrosine kinase inhibitor based
therapy. Curr. Pharm. Des. 10:1739-1744.
Coopman, P.J. and Mueller, S.C. (2006) The Syk tyrosine kinase:
a new negative regulator in tumor growth and progression.
Cancer Lett. 241:159-173.
Wang, X. et al. (2006) Syk is downstream of intercellular
adhesion molecule-1 and mediates human rhinovirus activation of
p38 MAPK in airway epithelial cells. J. Immunol. 177:6859-6870.
Humphrey, M.B. et al. (2005) Role of ITAM-containing adapter
proteins and their receptors in the immune system and bone.
Immunol. Rev. 208:50-65.
Sada, K. (2001) Structure and function of Syk protein-tyrosine
kinase. J. Biochem. (Tokyo). 130:177–186.
Shults, M.D. et al. (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.
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.
22
NOTES
23
Omnia® IP Kinase Assay Summary
24
Rev. A1
10/12/07
PR418