CycLex® Casein Kinase2 (CK2) Assay/Inhibitor Screening kit
Code No: CY-1170
Background:
Protein kinase CK2 is a ubiquitous and pleiotropic serine/threonine protein kinase, which appears to
interact with different signaling pathways and therefore represents the prototype of a multifunctional
protein kinase. The holoenzyme is generally composed of two catalytic (alpha and/or alpha') and two
regulatory (beta) subunits (1-3). Although the beta subunits deeply affect many properties of CK2, both
the free alpha/alpha' catalytic subunits and the holoenzyme are constitutively active. The enzyme is
highly expressed in most cancers (4) and this higher expression has been tentatively correlated with the
involvement of CK2 in the promotion of specific phases of the cell cycle (5). Unlike the majority of
protein kinases, which are tightly regulated enzymes, CK2 is endowed with high constitutive activity, a
feature that is suspected to underlie its oncogenic potential (6, 7) and possible implication in viral
infections. This makes CK2 an attractive target for anti-neoplastic and antiviral drugs.
Experimental studies suggest that dysregulated expression of the alpha subunit of CK2 imparts an
oncogenic potential in the cells such that in cooperation with certain oncogenes (8, 9), it produces a
profound enhancement of the tumor phenotype. Recent studies have provided evidence that
overexpression of CK2 in tumor cells is not simply a reflection of tumor cell proliferation alone but
additionally may reflect the pathobiological characteristics of the tumor. Of considerable interest is the
possibility that CK2 dysregulation in tumors may influence the apoptotic activity in those cells (10-12).
Approaches to interfering with the CK2 signal may provide a useful means for inducing tumor cell death
(13).
Measurement of CK2 activity
The protocol generally regarded as most sensitive for the quantitative measurement of CK2 activity
involves incubation of the CK2 sample with substrate (either a natural or synthetic polypeptide such as
CK2 substrate peptide; RRRDDDSDDD), in the presence of Mg2+ and 32P-labeled ATP. The reaction is
terminated by "spotting" a sample onto a filter paper disc, followed by immersion in acid to precipitate
the radiolabeled product. The filter papers are then washed extensively to remove unincorporated
radiolabel and the radioactivity is counted. While sensitive, this method is labor-intensive, generates
hazardous radioactive waste and depends on a radioisotope with a short half-life. It is particularly
unsuitable when kinase assays are only performed on an infrequent basis. The CycLex® CK2
Assay/Inhibitor Screening Kit uses a peroxidase coupled anti-phospho-p53 serine46 monoclonal
antibody as a reporter molecule in a 96-well ELISA format. This assay provides a non-isotopic, sensitive,
and specific method to measure CK2 activity.
Kit Content:
All samples and standards should be assayed in duplicate. The following components are supplied and
are sufficient for the one 96-wells microtiter plate kit.
Microplate: One microplate supplied ready to use, with 96 wells (12 strips of 8-wells) in a foil, zip-lock
bag with a desiccant pack. Wells are coated with recombinant full-length p53 as a substrate for CK2.
10X Wash Buffer: One 100 mL bottle of 10X buffer containing 2%Tween®-20.
Kinase Buffer: One 20 mL bottle of 1X buffer, used for Kinase Reaction Buffer and sample dilution.
20X ATP: Lyophilized ATP Na2 salt. Reconstitute contents of vial with 1 mL of H2O. Mix gently until
dissolved. The final concentration of the ATP stock solution is 2 mM ATP. The ATP solution can be
stored in small aliquots (e.g. 100 μL) at -20°C. The 2 mM ATP stock solution must be diluted to 100 μM
in Kinase Reaction Buffer immediately before use.
HRP conjugated Detection Antibody: One bottle containing 12 mL of HRP (horseradish peroxidase)
conjugated anti-phospho-p53 S46 (TK-4D4) antibody. Ready-to-use.
Substrate Reagent: 12 mL of the chromogenic substrate, tetra-methylbenzidine (TMB). Ready-to-use.
Stop Solution: 12 mL of 0.5 N H2SO4. Ready-to-use.
Related Products:
* CK2 (alpha/beta) Positive control: Cat# CY-E1170-1
* CK2 (alpha’/beta) Positive control: Cat# CY-E1170-2
* Anti-phospho-p53 S46 monoclonal antibody (TK-4D4): CY-M1022: Available soon
References:
1. Lozeman, F.J., Litchfield, D.W., Piening, C., Takio, K., Walsh, K.A. and Krebs, E.G. (1990) Isolation
and characterization of human cDNA clones encoding the α and α´ subunits of CK2. Biochemistry 29,
8436–8447
2. Litchfield, D.W., Lozeman, F.J., Piening, C., Sommercorn, J., Takio, K., Walsh, K.A. and Krebs, E.G.
(1990) Subunit structure of CK2 from bovine testis: demonstration that the α and α´ subunits are
distinct polypeptides. J. Biol. Chem. 265, 7638–7644
3. Maridor, G., Park, W., Krek, W. and Nigg, E.A. (1991) CK2. cDNA sequences, developmental
expression and tissue distribution of mRNAs for α, α´ and β subunits of the chicken enzyme. J. Biol.
Chem. 266, 2362–2368
4. Munstermann, U., Fritz, G., Seitz, G., Lu, Y.P., Schneider, H.R. and Issinger, O.-G. (1990) CK2 is
elevated in solid human tumors and rapidly proliferating non-neoplastic tissue. Eur. J. Biochem. 189,
251–257
5. Pepperkok, R., Lorenz, P., Ansorge, W. and Pyerin, W. (1994) CK2 is required for transition of G0/G1,
early G1, and G1/S phases of the cell cycle. J. Biol. Chem. 269, 6986–6991
6. Landesman-Bollag, E., Romieu-Mourez, R., Song, D.H., Sonenshein, G.E., Cardiff, R.D. and Seldin,
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Heparin (ng/ml)
Relative intensity (% of control)
CKIIα+CKIIβ
CKIIα'+CKIIβ
IC50;
CKIIα+CKIIβ: 65ng/ml
CKIIα’+CKIIβ: 20 ng/ml
CK2 Assay/Inhibitor Screening Kit Users Manual
Cat# CY-1170
4 H Constitution Way · Woburn, MA 01801 · Phone: 1.800.200.5459 · Fax: 781-939-6963 · www.mblintl.com
14 Version #040611-1
D.C. (2001) Protein kinase CK2 in mammary gland tumorigenesis. Oncogene 20, 3247–3257
7. Seldin, D.C. and Leder, P. (1995) CK2 alpha transgene-induce murine lymphoma: relation to
theileriosis in cattle. Science 267, 894–897
8. Landesman-Bollag, E., Channavajhala, P.L., Cardiff, R.D. and Seldin, D.C. (1998) p53 deficiency and
mis-expression of protein kinase CK2a collaborate in the development of thymic lymphomas in mice.
Oncogene 16, 2965–2974
9. Channavajhala, P. and Seldin, D.C. (2002) Functional interaction of protein kinase CK2 and c-Myc in
lymphomagenesis. Oncogene 21, 5280–5288
10. Sayed, M., Pelech, S., Wong, C., Marotta, A. and Salh, B. (2001) Protein kinase CK2 is involved in
G2 arrest and apoptosis following spindle damage in epithelial cells. Oncogene 20, 6994–7005
11. Desagher, S., Osen-Sand, A., Montessuit, S., Magnenat, E., Vilbois, F., Hochmann, A., Journot, L.,
Antonsson, B. and Martinou, J.C. (2001) Phosphorylation of bid by casein kinases I and II regulates
its cleavage by caspase 8. Mol. Cell 8, 601–611
12. Li, P., Li, J., Muller, E., Otto, A., Dietz, R. and von Harsdorf, R. (2002) Phosphorylation by protein
kinase CK2. A signaling switch for the caspase-inhibiting protein ARC. Mol. Cell 10, 247–258
13. Wang, H., Davis, A., Yu, S. and Ahmed, K. (2001) Response of cancer cells to molecular interruption
of the CK2 signal. Mol. Cell. Biochem. 227, 167–174
Precautions:
• Store the CK2 Positive Control enzyme at below -70°C and the ATP at -20°C when not in use. Store
all other components at 4°C. Do not expose reagents to excessive light. Avoid freeze/thaw cycles.
• Allow all the components to come to room temperature before use.
• All microplate strips that are not immediately required should be returned to the zip-lock pouch, which
must be carefully resealed to avoid moisture absorption.
• Do not use kit components beyond the indicated kit expiration date.
• Use only the microtiter wells provided with the kit.
• Rinse all detergent residue from glassware.
• Use deionized water of the highest quality.
• Do not mix reagents from different kits.
• The buffers and reagents used in this kit contain sodium Kathon-CG as a preservative. Care should be
taken to avoid direct contact with these reagents.
• Do not mouth pipette or ingest any of the reagents.
• Do not smoke, eat, or drink when performing the assay or in areas where samples or reagents are
handled.
• Human samples may be contaminated with infectious agents. Do not ingest, expose to open wounds or
breathe aerosols. Wear protective gloves and dispose of biological samples properly.
• Dispose of tetra-methylbenzidine (TMB) containing solutions in compliance with local regulations.
• CAUTION: Sulfuric Acid is a strong acid. Wear disposable gloves and eye protection when
handling Stop Solution.
Intended Use:
For Research use only. Not for use in diagnostic procedure.
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Apoptosis
Autophagy & Proteolysis
Cancer & Cell Proliferation
Cell Surface Antigens
Cellular Stress
Cytokines & Growth Factors
Epitope Tags
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In Vitro Diagnostics
Inflammation & Immunology
Molecular Biology/Genetics
Neuroscience
Signal Transduction
Solute Carrier
Virology
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