Recombinant Human Creatine Kinase MM/CKMM Protein, CF

Catalog # Availability Size / Price Qty
9070-CK-050
R&D Systems Recombinant Proteins and Enzymes
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Recombinant Human Creatine Kinase MM/CKMM Protein, CF Summary

Product Specifications

Purity
>95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
Endotoxin Level
<1.0 EU per 1 μg of the protein by the LAL method.
Activity
Measured by its ability to phosphorylate creatine. The specific activity is >1,900 pmol/min/μg, as measured under the described conditions.
Source
E. coli-derived human Creatine Kinase MM/CKMM protein
Pro2-Lys381, with N-terminal Met and 6-His tag
Accession #
N-terminal Sequence
Analysis
Met
Predicted Molecular Mass
44 kDa
SDS-PAGE
43 kDa, reducing conditions

Product Datasheets

Carrier Free

What does CF mean?

CF stands for Carrier Free (CF). We typically add Bovine Serum Albumin (BSA) as a carrier protein to our recombinant proteins. Adding a carrier protein enhances protein stability, increases shelf-life, and allows the recombinant protein to be stored at a more dilute concentration. The carrier free version does not contain BSA.

What formulation is right for me?

In general, we advise purchasing the recombinant protein with BSA for use in cell or tissue culture, or as an ELISA standard. In contrast, the carrier free protein is recommended for applications, in which the presence of BSA could interfere.

9070-CK

Formulation Supplied as a 0.2 μm filtered solution in Tris, NaCl and TCEP.
Shipping The product is shipped with polar packs. Upon receipt, store it immediately at the temperature recommended below.
Stability & Storage: Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 6 months from date of receipt, -20 to -70 °C as supplied.
  • 3 months, -20 to -70 °C under sterile conditions after opening.

Assay Procedure

Materials
  • Universal Kinase Activity Kit (Catalog # EA004)
  • 10X Assay Buffer (supplied in kit):  250 mM HEPES, 1.5 M NaCl, 100 mM MgCl2, 100 mM CaCl2, pH 7.0
  • Recombinant Human Creatine Kinase MM/CKMM (rhCKM) (Catalog # 9070-CK)
  • Creatine (Sigma, Catalog # C3630), 50 mM stock in deionized water
  • 96-well Clear Plate (Catalog # DY990)
  • Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
  1. Prepare 1X Assay Buffer by diluting 10X stocks 10-fold with deionized water.
  2. Dilute 1 mM Phosphate Standard provided by the Universal Kinase Activity Kit by adding 40 µL of the 1 mM Phosphate Standard to 360 µL of 1X Assay Buffer for a 100 µM stock. This is the first point of the standard curve.
  3. Complete the standard curve by performing six one-half serial dilutions of the 100 µM Phosphate stock using 1X Assay Buffer. The standard curve has a range of 0.078 to 5 nmol per well.
  4. Prepare a reaction mixture containing 0.4 mM ATP (supplied in kit) and 8 mM Creatine in 1X Assay Buffer.
  5. Dilute rhCKM to 6.667 ng/µL in 1X Assay Buffer.
  6. Dilute Coupling Phosphatase 4 (supplied in kit) to 10 µg/mL in 1X Assay Buffer.
  7. Load 50 µL of each dilution of the standard curve into a plate. Include a curve blank containing 50 μL of 1X Assay Buffer.
  8. Load 15 µL of the 6.667 ng/µL rhCKM into empty wells of the same plate as the curve. Include a Control containing 15 µL of 1X Assay Buffer.
  9. Add 10 µL of 10 µg/mL Coupling Phosphatase 4 to wells containing enzyme and control, excluding the standard curve.
  10. Add 25 µL of reaction mixture to the wells, excluding the standard curve.
  11. Cover the plate with a plate sealer and incubate at room temperature for 10 minutes.
  12. Add 30 µL of the Malachite Green Reagent A to all wells. Mix briefly.
  13. Add 100 µL of deionized water to all wells. Mix briefly.
  14. Add 30 µL of the Malachite Green Reagent B to all wells. Mix and incubate for 20 minutes at room temperature.
  15. Read plate at 620 nm (absorbance) in endpoint mode.
  16. Calculate specific activity:

     Specific Activity (pmol/min/µg) =

Phosphate released* (nmol) x (1000 pmol/nmol)
Incubation time (min) x amount of enzyme (µg) x coupling rate**

     *Derived from the phosphate standard curve using linear or 4-parameter fitting and adjusted for Control.
     ** The coupling rate is 0.475 under these conditions.

Per Reaction:

  • rhCKM: 0.1 µg
  • Coupling Phosphatase 4: 0.1 µg
  • Creatine: 4 mM
  • ATP: 0.2 mM
Reconstitution Calculator

Reconstitution Calculator

The reconstitution calculator allows you to quickly calculate the volume of a reagent to reconstitute your vial. Simply enter the mass of reagent and the target concentration and the calculator will determine the rest.

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Background: Creatine Kinase MM/CKMM

Creatine kinase (CK) catalyzes the conversion of creatine to phosphocreatine (PCr) by consuming adenosine triphosphate (ATP) and generating adenosine diphosphate (ADP). CK reaction is reversible and thus ATP can be generated from PCr and ADP (1). In tissues and cells that consume ATP rapidly, especially skeletal muscle, but also brain, photoreceptor cells of the retina, hair cells of the inner ear, spermatozoa and smooth muscle, PCr serves as an energy reservoir for the rapid buffering and regeneration of ATP in situ, as well as for intracellular energy transport by the PCr shuttle or circuit (2). Clinically, creatine kinase is assayed in blood tests as a marker of myocardial infarction (heart attack), rhabdomyolysis (severe muscle breakdown), muscular dystrophy, autoimmune myositides and acute renal failure. Creatine kinase M (CKM), especially, is a cytoplasmic enzyme and is an important serum marker for myocardial infarction. It acts as a homodimer (MM type) in striated muscle as well as in other tissues, and forms a heterodimer (MB type) with CKB, a brain isozyme, in the heart (3). The recombinant human CKM is in the MM type and its activity is measured using a phosphatase-coupled method (4).

References
  1. Trask, R.V. et al. (1988) J. Biol. Chem. 263:17142.
  2. Wallimann, T. et al. (1992) Biochem, J. 281:21.
  3. Wallimann, T. (1994) Curr. Biol.4:42.
  4. Wu, Z.L. (2011) PLoS One 6:e23172.
Long Name
Creatine Kinase, Muscle
Entrez Gene IDs
1158 (Human); 12715 (Mouse); 24265 (Rat)
Alternate Names
CKM; CKMM; CKMMcreatine kinase M-type; Creatine Kinase Isoenzyme; Creatine kinase M chain; Creatine Kinase MM; creatine kinase, muscle; creatine kinase-M; EC 2.7.3; EC 2.7.3.2; M-CK

Citation for Recombinant Human Creatine Kinase MM/CKMM Protein, CF

R&D Systems personnel manually curate a database that contains references using R&D Systems products. The data collected includes not only links to publications in PubMed, but also provides information about sample types, species, and experimental conditions.

1 Citation: Showing 1 - 1

  1. Acetylation of muscle creatine kinase negatively impacts high-energy phosphotransfer in heart failure
    Authors: MA Walker, J Chavez, O Villet, X Tang, A Keller, JE Bruce, R Tian
    JCI Insight, 2021;6(3):.
    Species: Human
    Sample Types: Tissue Homogenates
    Applications: Bioassay

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