Creatine Kinase Assay Kit

Catalog No. ABIN1000303

Product Details

Target: Creatine Kinase

Application: Activity Assay (AcA)

Sample Type: Plasma, Serum

Specificity: 5 U/L

Characteristics: Sensitive and accurate. Detection range: 5 to 300 U/L creatine kinase in 96-well plate assay.
Convenient. The procedure involves adding a single working reagent, and reading the optical density at 20 min and 40 min at room temperature or 37°C.
High-throughput. Can be readily automated as a high-throughput 96- well plate assay for thousands of samples per day.

Components: Assay Buffer: 12 mL. Substrate Solution: 1.0 mL. Enzyme Mix: 120 µL. Calibrator: 150 µL.

Material not included: Pipeting (multi-channel) devices. Clear-bottom 96-well plates (e.g. Corning Costar) and plate reader.

Application Details

Application Notes: Direct Assays: CK in serum, plasma and other biological samples.
Pharmacology: effects of drugs on CK activity.

Comment:

This assay is based on an enzyme-catalyzed kinetic reaction. Addition of Reconstituted Reagent should be quick and mixing should be brief but thorough. Use of multi-channel pipettor is recommended.

Protocol: 1. Calibrator: transfer 110 µL water and (10 µL Calibrator + 100 µL water) into separate wells of a clear bottom 96-well plate. Samples: transfer 10 µL samples into separate wells. Add 100 µL Reconstituted Reagent and tap plate to mix.
2. Reaction. Incubate at room temperature or 37°C. CK is fully activated within 20 min by glutathione provided in the Substrate Solution. Read OD340nm at 20 min and again at 40 min.

Sample Preparation:

Use non-hemolyzed samples. Samples should be assayed within 4 hours of blood collection if they remain at room temperature, or within 12 hours if stored at 4°C. Samples can be stored at -80°C and should be thawed only once. If turbidity is observed, centrifuge sample and use clear supernatant for assay. Reagent Reconstitution. Bring all components to room temperature. For each reaction well, mix 10 µL Substrate Solution, 100 µL Assay Buffer and 1 µL Enzyme Mix. Fresh reconstitution is recommended. If the assay is to be carried out at 37°C, warm up the Reconstituted Reagent at 37°C.

Restrictions: For Research Use only

Handling

Storage: -20 °C

References

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Target Details

Target: Creatine Kinase

Background: Quantitative determination of creatine kinase activity at 340nm.
Procedure: 5 min.

Creatine Kinase (CK), also known as creatine phosphokinase (CPK), is an enzyme (EC 2.7.3.2) expressed predominantly in skeletal muscle, smooth muscle and the brain. The CK enzyme consists of two subunits, which can be either B (brain type) or M (muscle type), and hence three different isoenzymes: CK-MM, CK-BB and CK-MB. CK catalyzes the conversion of creatine to phosphocreatine, consuming adenosine triphosphate (ATP) and generating adenosine diphosphate (ADP) and the reverse reaction. CK is often determined routinely in emergency patients with chest pain and acute renal failure. Elevation of CK is an indication of damage to muscle and has been associated with injury, rhabdomyolysis, myocardial infarction, myositis, myocarditis, malignant hyperthermia and neuroleptic malignant syndrome, etc. Lower levels can be an indication of alcoholic liver disease and rheumatoid arthritis. Simple, direct and automation-ready procedures for measuring CK activity are very desirable. This Creatine Kinase Assay Kit is based on enzyme coupled reactions in which creatine phosphate and ADP is converted to creatine and ATP by CK, the generated ATP is used to phosphorylate glucose by hexokinase to generate glucose-6-phosphate, which is then oxidized by NADP in the presence of glucose-6-phosphate dehydrogenase. The produced NADPH, measured at 340 nm, is proportionate to the CK activity in the sample.