Acetokinase (ACK) Activity Assay Kit (UV Micro Method) - BioReagent, high purity

Cat. No.: A1501276

GRADE & PURITY BioReagent

Descripción general

Acetate Kinase (ACK) is primarily found in microorganisms. It catalyzes the conversion of acetate and ATP to acetyl phosphate and ADP, serving as a key enzyme in bacterial carbon and energy metabolism, and plays a central role particularly in the methanogenesis metabolism of archaea.

Assay Principle
ACK catalyzes the synthesis of Acetyl Phosphate and ADP from Sodium Acetate and ATP. Pyruvate Kinase then catalyzes the conversion of ADP and Phosphoenolpyruvate (PEP) to ATP and Pyruvate. Subsequently, Lactate Dehydrogenase catalyzes the reduction of Pyruvate by NADH to produce Lactate and NAD⁺. The rate of oxidation of NADH to NAD⁺, measured by the decrease in absorbance at 340 nm, reflects ACK activity.

Component	48T	96T	Storage
Extraction Buffer	60 mL	60 mL×2	2-8℃
ReagentⅠ	15 mL	30 mL	2-8℃
ReagentⅡ	1EA	2EA	-20℃. Store in the dark.
Reagent III	25 μL	50 μL

Note: It is recommended to perform a pilot experiment with 2-3 samples expected to have significant differences before formal testing.

Required Materials and Equipment (Not Provided)

Microplate reader or UV spectrophotometer (capable of measuring absorbance at 340 nm)

96-well UV plate or micro quartz cuvette

Adjustable pipettes and tips

Constant temperature water bath

Ice maker

Centrifuge

Deionized water

Homogenizer (for tissue samples)

Reagent Preparation

Extraction Buffer: Ready-to-use. Equilibrate to room temperature (RT) before use. Store at 4°C.
Caution: Extraction Buffer is toxic and has a pungent odor. It is recommended to handle it within a fume hood.

Reagent Ⅰ: Ready-to-use. Equilibrate to RT before use. Store at 4°C.

Working Reagent Ⅱ: Prepare immediately before use. For one vial of Reagent Ⅱ, add 11 mL of Reagent Ⅰ and 19.8 μL of Reagent III. Mix thoroughly to dissolve. Prepare fresh for each use. Can be stored protected from light at -20°C for one month.

Reagent Ⅲ: Ready-to-use. Equilibrate to RT before use. Store at 4°C protected from light.

Sample Preparation

*Note: The use of fresh samples is recommended. If not used immediately, samples can be stored at -80°C for up to one month. Control the temperature and time during thawing. If thawed at room temperature, complete the process within 4 hours.*

1.Tissues: Weigh approximately 0.1 g of sample. Add 1 mL of Extraction Buffer and homogenize on ice. Centrifuge the homogenate at 15,000 g, 4°C for 10 min. Collect the supernatant and keep it on ice for assay.

2.Cells or Bacteria: Collect 5 million cells or bacteria by centrifugation. Wash the pellet with cold PBS, centrifuge, and discard the supernatant. Add 1 mL of Extraction Buffer. Disrupt the cells/bacteria by sonication on ice (200W power, pulse 3s on/10s off, repeat 30 times). Centrifuge the lysate at 15,000 g, 4°C for 10 min. Collect the supernatant and keep it on ice for assay.

3.Serum (Plasma) or other liquid samples: Assay directly. If the solution is turbid, centrifuge first and use the supernatant for assay.

Note: To determine protein concentration, Aladdin's BCA Protein Quantification Kit (B665595) or Ready-to-Use BCA Protein Quantification Kit (R1491648) is recommended.

Assay Procedure

1.Preheat the microplate reader or spectrophotometer for 30 min. Set the wavelength to 340 nm. Zero the spectrophotometer with deionized water.

2.Pre-warm a sufficient volume of the prepared Working Reagent Ⅱ at 37°C (for mammalian samples) or 25°C (for other species) for 5 minutes. Use immediately.

3.Assay Setup (perform in a 96-well UV plate or micro quartz cuvette):

Reagent	Test Well (μL)
Sample	20
Working Reagent Ⅱ	180

Mix thoroughly immediately after addition. Measure the absorbance at 340 nm at 10 seconds (A₁) and again at 190 seconds (A₂). Calculate ΔA = A₁ - A₂.

Note: It is advised to run a pilot test with 2-3 samples showing expected significant variation beforehand. If ΔA is less than 0.05, consider increasing the sample volume or extending the reaction time to 10 or 20 minutes before measurement. If ΔA is greater than 1.0, dilute the sample further with Extraction Buffer (multiply the result by the dilution factor) or reduce the amount of sample used for extraction.

Result Calculation

Note: Both the derived and simplified calculation formulas are provided and are equivalent. The simplified formulas (in bold) are recommended for final calculation.

1. Calculation for 96-Well UV Plate

General Parameters for 96-Well Plate:

ε (NADH molar extinction coefficient) = 6.22 × 10³ L/mol/cm

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

Vₜₒₜₐₗ (Total reaction volume) = 0.0002 L (200 μL)

Vₛₐₘₚₗₑ (Sample volume in reaction) = 0.02 mL (20 μL)

T (Reaction time) = 3 min

Vₛₐₘₚₗₑₜₒₜₐₗ (Total extraction volume) = 1 mL

Cpr (Sample protein concentration, mg/mL)

W (Sample mass, g)

500 (Cell/Bacteria count in millions: 5 × 10⁶)

1.1 Based on Sample Protein Concentration:

Definition: One unit of activity is defined as the amount of enzyme that consumes 1 nmol of NADH per minute per mg of protein.

Calculation:
ACK Activity (U/mg prot) = [ΔA × Vₜₒₜₐₗ ÷ (ε × d) × 10⁹] ÷ (Vₛₐₘₚₗₑ × Cpr) ÷ T
Simplified Formula: ACK (U/mg prot) = 1072 × ΔA ÷ Cpr

1.2 Based on Sample Mass:

Definition: One unit of activity is defined as the amount of enzyme that consumes 1 nmol of NADH per minute per gram of fresh sample.

Calculation:
ACK Activity (U/g fresh weight) = [ΔA × Vₜₒₜₐₗ ÷ (ε × d) × 10⁹] ÷ (W × Vₛₐₘₚₗₑ / Vₛₐₘₚₗₑₜₒₜₐₗ) ÷ T
Simplified Formula: ACK (U/g fresh weight) = 1072 × ΔA ÷ W

1.3 Based on Bacterial or Cell Density:

Definition: One unit of activity is defined as the amount of enzyme that consumes 1 nmol of NADH per minute per 10⁴ cells/bacteria in the reaction system.

Calculation (for 5 million cells in 1 ml extract):
ACK Activity (U/10⁴) = [ΔA × Vₜₒₜₐₗ ÷ (ε × d) × 10⁹] ÷ (500 × Vₛₐₘₚₗₑ / Vₛₐₘₚₗₑₜₒₜₐₗ) ÷ T
Simplified Formula: ACK (U/10⁴) = 2.144 × ΔA

1.4 Based on Liquid Volume:

Definition: One unit of activity is defined as the amount of enzyme that consumes 1 nmol of NADH per minute per milliliter of sample.

Calculation:
ACK Activity (U/mL) = [ΔA × Vₜₒₜₐₗ ÷ (ε × d) × 10⁹] ÷ Vₛₐₘₚₗₑ ÷ T
Simplified Formula: ACK (U/mL) = 1072 × ΔA

2. Calculation for Micro Quartz Cuvette
Use the formulas above but adjust the light path *d* from 0.5 cm to 1.0 cm.

Precautions

1. Keep samples and all reagents on ice during the assay procedure to prevent denaturation and loss of activity.

2. The temperature of the reaction mixture must be maintained at 37°C or 25°C. When using a cuvette, a small beaker filled with deionized water pre-warmed to 37°C or 25°C (placed in a water bath) can be used to hold the cuvette and maintain temperature during the reaction.

3. This product is for scientific research use only. It is not intended for clinical diagnosis. For your safety and health, please wear a lab coat and disposable gloves during operation.

Almacenamiento y envío

Condiciones de almacenamiento de almacenamiento

Protected from light,Store at -20°C

Enviado en

Ice chest + Ice pads

Estabilidad y almacenamiento

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