Creatin Kinase (CK) Activity Assay Kit (UV Micro Method) - BioReagent, high purity

Cat. No.: C1501222

GRADE & PURITY BioReagent

Overview

Creatine Kinase (CK) is primarily found in tissues such as the heart, muscle, and brain. It reversibly catalyzes the transphosphorylation reaction between creatine and ATP, playing a vital role in energy transfer, muscle contraction, and ATP regeneration. It is a crucial clinical indicator for diagnosing heart and brain diseases.

Assay Principle
CK catalyzes the conversion of Phosphocreatine and ADP to Creatine and ATP. Hexokinase then catalyzes the reaction of ATP with Glucose to form Glucose-6-Phosphate (G6P). Subsequently, Glucose-6-Phosphate Dehydrogenase (G6PDH) catalyzes the oxidation of G6P with NADP⁺ to generate NADPH, leading to an increase in absorbance at 340 nm.

Component	100T	Storage
Extraction Buffer	100 mL	2-8℃
Reagent 1	1EA	2-8℃. Store in the dark.
Reagent 2	10 mL	2-8℃

• Reagent 1: Powder in one bottle. Store at 4°C protected from light. Dissolve in 10 mL distilled water before use.

• Working Solution: Prepare immediately before use by mixing Reagent 1 and Reagent 2 at a 1:1 ratio. Incubate the Working Solution at 37°C for 2 minutes prior to use.

Required Materials and Equipment (Not Provided)
Balance, refrigerated centrifuge, constant temperature water bath, microplate reader, 96-well plate, and distilled water.

Crude Enzyme Extraction:

1. Tissue Samples: Homogenize the tissue on ice in Extraction Buffer at a ratio of 1:5-10 (w/v) (e.g., weigh ~0.1g tissue, add 1 mL Extraction Buffer). Centrifuge the homogenate at 10,000 g, 4°C for 15 min. Collect the supernatant for assay.

2. Serum Samples: assay directly.

Assay Procedure:

1. Preheat the microplate reader for at least 30 minutes. Set the wavelength to 340 nm.

2. Pipette 40 μl of sample and 60 μl of distilled water into a well of the 96-well plate. Add 100 μl of the pre-warmed (37°C) Working Solution. Mix immediately and record the initial absorbance (A₁) and the absorbance after exactly 1 minute (A₂) at 37°C. Calculate ΔA = A₂ - A₁.

CK Enzyme Activity Calculation:

General Parameters:

• ε (NADPH molar extinction coefficient) = 6220 L/mol/cm

• d (Light path for 96-well plate) = 0.5 cm

• Vₜₒₜₐₗ (Total reaction volume) = 0.2 mL (200 μL)

• Vₛₐₘₚₗₑ (Sample volume in reaction) = 0.04 mL (40 μL)

• T (Reaction time) = 1 min

• Cpr (Sample protein concentration, mg/mL)

• W (Sample mass, g)

• Vₛₐₘₚₗₑₜₒₜₐₗ (Total extract volume) = Assumed 1 mL for tissue calculations

1. Based on Tissue Protein Content:

• Definition: One unit of activity is defined as the amount of enzyme that generates 1 nmol of NADPH per minute per mg of protein at 37°C, pH 7.0.

• Calculation:
  CK Activity (nmol/min/mg prot) = [ΔA / (ε × d)] × Vₜₒₜₐₗ ÷ (Vₛₐₘₚₗₑ × Cpr) ÷ T
  Simplified Formula: CK (nmol/min/mg prot) = 1608 × ΔA ÷ Cpr

2. Based on Tissue Sample Mass:

• Definition: One unit of activity is defined as the amount of enzyme that generates 1 nmol of NADPH per minute per gram of fresh tissue at 37°C, pH 7.0.

• Calculation:
  CK Activity (nmol/min/g fresh weight) = [ΔA / (ε × d)] × Vₜₒₜₐₗ ÷ (Vₛₐₘₚₗₑ / Vₛₐₘₚₗₑₜₒₜₐₗ × W) ÷ T
  Simplified Formula: CK (nmol/min/g fresh weight) = 1608 × ΔA ÷ W

3. Based on Serum:

• Definition: One unit of activity is defined as the amount of enzyme that generates 1 nmol of NADPH per minute per liter of serum at 37°C, pH 7.0.

• Calculation:
  CK Activity (nmol/min/L) = [ΔA / (ε × d)] × Vₜₒₜₐₗ ÷ Vₛₐₘₚₗₑ ÷ T
  Simplified Formula: CK (nmol/min/L) = 1608 × ΔA

Notes

1. Before formal assay, it is essential to perform a pilot test with 2-3 samples expected to have significant differences in activity.

2. The prepared Working Solution is stable at 4°C for 7 days. However, it is recommended to use it as soon as possible after preparation.

3. CK in serum is unstable. Determine the activity as soon as possible after sample collection. It can be stored protected from light at 4°C for up to 24 hours.

4. Sample protein content needs to be determined separately. A BCA Protein Assay Kit can be used for this purpose.

5. If the OD value is greater than 0.5, dilute the sample appropriately with Extraction Buffer and account for the dilution factor (D) in the calculation formulas (e.g., 1608 × ΔA × D ÷ Cpr).

Storage and Shipping

Storage

Store at 2-8°C,Protected from light

Shipped In

Wet ice

Stability And Storage

Each component has a shelf life of 6 months under corresponding storage conditions.