Creatine Kinase (CK) Activity Colorimetric Assay Kit

Details for Product No. ABIN1304379, Supplier: Log in to see
Antigen
Reactivity
General
3
1
1
1
Minimum Detection Limit
< 1 mU
Application
Activity Assay (AcA)
Options
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Supplier Product No.
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'Independent Validation' Badge
Antigen CK
Lot Number 3A26K07770
Method validated Activity Assay
Positive Control Myotube cells lysate (total intracellular CK)
Negative Control Blank was measured as background.
Notes

In our hands, ABIN1304379 performed very well in terms of sensitivity, precision and reproducibility.

Protocol
  • Incubate Human primary myotubes derived from an healthy subject and from a Limb Girdle Muscular Dystrophy type 2D (LGMD2D) patient (dystrophic) in 250µl of hypo-osmotic solutions (200 or 260mOsm) preheated at 37°C.
  • The hypo-osmotic solutions consists of a salt solution (5mM HEPES, 5mM KCl, 1mM MgCl2, 5mM NaCl, 1.2mM CaCl2, 1mM glucose) supplemented with 157mM (200mOsm) or 213mM (260mOsm) sucrose. Osmolarities are measured with an OM 801 Osmometer (Vogel).
  • After 20min at 37°C, collect the supernatants containing the released CK and store them at -80°C. To measure total intracellular CK, lyse cells by three freeze-thaw cycles. Store the cellular lysates at -80°C until CK analysis.
  • Measure released and intracellular CK with the Creatine Kinase Activity Colorimetric Assay Kit (antibodies-online, ABIN1304379, lot 3A26K07770) according to the manufacturer’s instructions.
  • Measure an NADH standard curve in end-point mode (figure panel A).
  • Measure the CK amount in a 96 well multiplate in a reaction volume of 100µl.
  • Monitor the accumulation of the reaction product in a kinetic mode on a Multiskan EX microplate reader (Thermo Scientific, 51118170), recording the OD450 every 1min for 60min (figure panel B).
  • Subtract a blank value from all readings.
  • Calculate the amount of creatine kinase in the samples measuring the slope of the straight line interpolating the linear part of the OD450 vs time curves, and comparing this value with the NADH standard curve (figure panel C).
  • Perform each experiment twice in sextuplicate. Error bars represent the SEM.
Experimental Notes
  • Figure panel B shows an example of the spectrophotometric traces recorded measuring the colorimetric reaction at 450nm. It is possible to obtain a reaction that proceeds in a linear manner, which allows an accurate determination of the initial velocity of the reaction by linear regression (R2 value=0.99).

  • Panel A reports the standard curve obtained with the NADH standard supplied with the kit.

  • The graph in panel C shows the percentage of CK release from myotubes of a healthy and a dystrophic subject, expressed as the ratio between the released and total (released+intracellular) CK. As expected, myotubes derived from the healthy subject released significantly less CK when exposed to hypo-osmotic conditions than dystrophic myotubes. Furthermore, the release of CK from the dystrophic myotubes increased by worsening the stress condition (i.e. by lowering the osmolarity of the solution).

  • Our results demonstrate that this kit may be confidently used in an in vitro functional test to assess the stability of the sarcolemma. This test may be useful for a preliminary evaluation of the efficacy of genetic or pharmacological approaches for the treatment of LGMD2D.

Validation Images
Activity Assay validation image for Creatine Kinase (CK) Activity Colorimetric Assay Kit (ABIN1304379) A standard curve obtained with the NADH standard supplied with ABIN1304379. B Example...
Sample Type Adherent Cell Culture, Cell Culture Cells, Cell Samples, Plasma, Tissue Samples
Detection Method Colorimetric
Characteristics Creatine Kinase (CK) Activity Colorimetric Assay Kit: Rapid, Sensitive & High-throughput Adaptable Assay to detect CK activity in serum, plasma, animal tissues & adherent & suspension cells etc.
Simple procedure, takes approx. 30-40 minutes, Sensitive and high-throughput adaptable, Kit contains all necessary reagents for measuring creatine kinase activity

Detection wavelength: 450 nm.
Components CK Assay Buffer
CK Substrate
ATP (Lyophilized)
CK Enzyme Mix (Lyophilized)
CK Developer (Lyophilized)
NADH Standard (Lyophilized)
Positive Control (Lyophilized)
Target Name (Antigen)
Background Creatine Kinase (CK) also known as creatine phosphokinase (CPK) and ATP: creatine N-phosphotransferase is a common cellular enzyme (EC 2.7.3.2). It catalyzes the reversible conversion of creatine and ATP into ADP and phosphocreatine. CK is widely expressed in various tissues and cell types, with highest activity in striated muscles, heart tissue and brain. CK consists of two subunits: M (muscle) and B (brain), and has three isoenzymes: CK-MM (skeleton muscle), CK-MB (cardiac muscle), and CK-BB (brain). Increased CK level is associated with many diseases such as myocardial infarction, muscular dystrophy, pulmonary infarction and brain tumors. Accurate measurement of CK is crucial for early diagnosis, prediction and therapeutic strategy. In
Application Notes The kit can detect creatine kinase activity less than 1 mU.
Comment

Further details regarding sample type:

  • Serum & plasma.
  • Animal tissues: muscle, brain, heart etc.
  • Cell culture: Adherent or suspension cells.

Restrictions For Research Use only
Storage -20 °C
Expiry Date 12 months
Supplier Images
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