Fructokinase (FRK) Activity Assay Kit (UV Colorimetric Method) - BioReagent, high purity

Cat. No.: F1505403

GRADE & PURITY BioReagent

Overview

  Fructokinase (FK, EC 2.7.1.4) regulates the interconversion between sucrose and starch and is involved in modulating plant metabolism and growth development.
  Fructokinase (FK) phosphorylates fructose to generate fructose-6-phosphate. This product is subsequently acted upon by a series of composite enzymes, reducing NADP+ to NADPH. The enzyme activity of fructokinase is determined by measuring the rate of increase in NADPH absorbance at 340 nm.

Component	50T	Storage
Extraction Buffer	60 mL	2-8℃
Reagent 1	40 mL	2-8℃
Reagent 2	1EA	-20℃
Reagent 3	1EA	-20℃
Reagent 4	1EA	2-8℃
Reagent 5	1EA	2-8℃

Reagent Preparation

• Reagent 2 (Powder, 1 vial):

  1. Before use, centrifuge at 8000 g, 4°C for 2 min to collect the powder at the bottom.

  2. Add 1.7 mL of distilled water to dissolve.

  3. The storage period is the same as the kit's expiry date.

• Reagent 3 (Liquid, 1 vial):

  1. Before use, centrifuge at 8000 g, 4°C for 2 min to collect the liquid at the bottom.

  2. Add 1.7 mL of distilled water to dissolve.

  3. The storage period is the same as the kit's expiry date.

• Reagent 4 (Powder, 1 vial):

  1. Before opening, ensure the powder is at the bottom of the vial.

  2. Add 17 mL of Reagent 1 to dissolve.

  3. The storage period is the same as the kit's expiry date.

• Reagent 5 (Liquid, 1 vial):

  1. Before use, centrifuge at 8000 g, 4°C for 2 min to collect the liquid at the bottom.

  2. Add 1.7 mL of distilled water to dissolve.

  3. The storage period is the same as the kit's expiry date.
     User-Prepared Instruments & Materials
     Mortar (homogenizer), ice bucket (ice maker), benchtop centrifuge, adjustable pipettes, water bath (oven, incubator, metal bath), 1 ml quartz cuvette, centrifuge tubes, UV spectrophotometer, distilled water (deionized water or ultrapure water is acceptable).

Sample Extraction

1. Tissue Samples: Weigh approximately 0.1 g of tissue, add 1 mL of Extraction Buffer, and homogenize on ice. Centrifuge at 12000 rpm, 4°C for 10 minutes. Collect the supernatant and keep it on ice for assay.
Note: If increasing the sample amount, use a ratio of 1:5 to 1:10 (tissue weight (g) : Extraction Buffer volume (mL)) for extraction.

2. Bacterial/Cell Samples: Collect bacteria or cells into a centrifuge tube by centrifugation and discard the supernatant. Take 5 million bacteria or cells, add 1 mL of Extraction Buffer, and disrupt using ultrasound on ice (power 20% or 200 W, ultrasonicate for 3 s, interval 10 s, repeat 30 times). Centrifuge at 12000 rpm, 4°C for 10 minutes. Collect the supernatant and keep it on ice for assay.
Note: If increasing the sample amount, use a ratio of 500-1000 (x10⁴ cells) : 1 (mL Extraction Buffer) for extraction.

3. Liquid Samples: Detect directly. If the sample is turbid, centrifuge and use the supernatant for detection.

Assay Procedure

1. Preheat the UV spectrophotometer for at least 30 minutes. Set the wavelength to 340 nm. Zero the instrument with distilled water.

2. Thaw all reagents to room temperature (25°C).

3. In a 1 mL quartz cuvette, add sequentially:

Reagent (μL)	Test Cuvette
Sample	70
Reagent 2	30
Reagent 3	430
Reagent 4	580

Mix well and incubate at 37°C for 5 minutes. 

4. Add:

Reagent (μL)	Test Cuvette
Reagent 5	30

5. Mix well. Immediately read the absorbance at 340 nm (A1), and then read again after 15 minutes (A2). Calculate ΔA = A2 - A1.

Notes:

1. If ΔA is close to zero, the reaction time can be appropriately extended to 30 minutes or longer before reading A2; or the sample volume V1 can be increased appropriately (with a corresponding decrease in Reagent 4 volume). The modified reaction time (T) and sample volume (V1) must be substituted into the calculation formula.

2. If the initial absorbance A1 is too high (e.g., >2, as in deeply pigmented plant leaves), appropriately reduce the sample volume V1 (with a corresponding increase in Reagent 4 volume). The modified V1 must be substituted into the calculation formula. Alternatively, add a small amount of activated carbon to the sample, mix, let stand for 5 min, then centrifuge at 12000 rpm, 4°C for 10 min, and use the supernatant for detection.

3. If the increasing trend is unstable, read the absorbance every 10 seconds and select a linear increasing period for calculation. The corresponding ΔA value should be substituted into the calculation formula.

FK Activity Calculation

1. Based on Sample Protein Concentration:

• Unit Definition: One unit of enzyme activity is defined as the generation of 1 nmol NADPH per minute per mg of protein.

• Formula:
  FK (nmol/min/mg prot) = [ΔA ÷ (ε × d) × V2 × 10⁹] ÷ (V1 × Cpr) ÷ T = 113.3 × ΔA ÷ Cpr

2. Based on Sample Fresh Weight:

• Unit Definition: One unit of enzyme activity is defined as the generation of 1 nmol NADPH per minute per gram of fresh tissue.

• Formula:
  FK (nmol/min/g fresh weight) = [ΔA ÷ (ε × d) × V2 × 10⁹] ÷ (W × V1 ÷ V) ÷ T = 113.3 × ΔA ÷ W

3. Based on Bacterial/Cell Density:

• Unit Definition: One unit of enzyme activity is defined as the generation of 1 nmol NADPH per minute per 10⁴ bacteria/cells.

• Formula:
  FK (nmol/min/10⁴ cell) = [ΔA ÷ (ε × d) × V2 × 10⁹] ÷ (500 × V1 ÷ V) ÷ T = 0.227 × ΔA

4. Based on Liquid Volume:

• Unit Definition: One unit of enzyme activity is defined as the generation of 1 nmol NADPH per minute per mL of liquid.

• Formula:
  FK (nmol/min/mL) = [ΔA ÷ (ε × d) × V2 × 10⁹] ÷ V1 ÷ T = 113.3 × ΔA

Parameter Description:

• ε: NADPH molar extinction coefficient, 6.22 × 10³ L/mol/cm

• d: Light path of the 1 mL quartz cuvette, 1 cm

• V: Volume of Extraction Buffer added, 1 mL

• V1: Volume of sample supernatant added, 0.07 mL

• V2: Total reaction volume, 0.74 mL = 7.4 × 10⁻⁴ L

• T: Reaction time, 15 min

• W: Sample mass, g

• 500: Cell number, in units of 10⁴

• Cpr: Protein concentration of the supernatant, mg/mL; Aladdin BCA Protein Quantification Kit (B665595) or Ready-to-Use BCA Protein Quantification Kit (R1491648) are recommended.

Precautions
It is recommended to first select 1-3 samples with significant differences (e.g., different types or groups) for preliminary experiments to familiarize yourself with the procedure. Determine or adjust the sample concentration based on the preliminary results to prevent unnecessary waste of samples or reagents.

Storage and Shipping

Storage

Store at -20°C

Shipped In

Ice chest + Ice pads

Stability And Storage

Each component has a shelf life of 3 months under corresponding storage conditions.