3-Phosphoglycerate Kinase (PGK) Activity Assay Kit (UV Colorimetric Method) - BioReagent, high purity

Cat. No.: P1505407

GRADE & PURITY BioReagent

Overview

  3-Phosphoglycerate kinase (PGK) is a key enzyme in glycolysis, widely present in animals, plants, and microorganisms. It catalyzes the reaction of 3-phosphoglycerate and ATP to produce 1,3-bisphosphoglycerate. The latter, under the action of glyceraldehyde-3-phosphate dehydrogenase and NADH, produces glyceraldehyde-3-phosphate and NAD+. The activity of 3-phosphoglycerate kinase (PGK) is determined by measuring the decrease in NADH.

Component	50T	Storage
Extraction Buffer	50 mL	2-8℃. Store in the dark.
Reagent 1	1EA	-20℃. Store in the dark.
Reagent 2	3EA	2-8℃
Reagent 3	1EA	-20℃
Reagent 4	35 mL	2-8℃
Reagent 5	1EA	-20℃

Reagent Preparation:

• Reagent 1 (Powder, 1 vial):

  1. Before opening, ensure the powder is at the bottom of the vial (tap manually if needed).

  2. Add 2.2 mL of distilled water to dissolve. The dissolved reagent can be aliquoted and stored at -20°C.

• Reagent 2 (Powder, 3 vials):

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

  2. Add 0.4 mL of distilled water to dissolve. The dissolved reagent can be aliquoted and stored at -20°C (use within one month after dissolution).

• 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 1.1 mL of distilled water to dissolve. The dissolved reagent can be aliquoted and stored at -20°C.

• Reagent 5 (Powder, 1 vial):

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

  2. Add 1.1 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, homogenize on ice, and then centrifuge at 12000 rpm, 4°C for 5 minutes. Collect the supernatant 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 approximately 5 million bacteria or cells, add 1 mL of Extraction Buffer, and disrupt using ultrasound on ice (power 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.

Assay Procedure

1. Preheat the UV spectrophotometer for 30 minutes. Set the wavelength to 340 nm and the temperature to 25°C. Zero the instrument with distilled water.

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

3. In a 1 mL quartz cuvette (1 cm light path), add sequentially:

Reagent (μL)	Test Tube
Sample	80
Reagent 1	40
Reagent 2	20
Reagent 3	20
Reagent 4	600

Mix well and incubate at room temperature (25°C) for 10 minutes.

4. Add 

Reagent (μL)	Test Tube
Reagent 5	20

5. Mix gently. At room temperature (25°C), read the absorbance at 340 nm at 30 seconds (A1) and then again after 10 minutes (A2). Calculate ΔA = A1 - A2.

Notes:

1. If ΔA is close to zero, the reaction time can be appropriately extended to 20 minutes before reading A2. The modified reaction time must be substituted into the calculation formula. Alternatively, increase the sample volume appropriately (e.g., 100 μL, with a corresponding decrease in Reagent 4 volume). The modified sample volume must be substituted into the calculation formula.

2. If the decreasing trend is unstable, read the absorbance every 20 seconds and select a linear decreasing period for calculation. The corresponding ΔA value should be substituted into the calculation formula.

3. If the initial absorbance A1 is too high (e.g., >2, as in deeply pigmented plant leaves), appropriately reduce the sample volume. The modified sample volume 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.

4. If ΔA is greater than 0.5, reduce the reaction time (e.g., to 5 min) or reduce the sample volume (e.g., to 20 μL). The modified reaction time (T) and sample volume (V1) must be substituted into the calculation formula.

PGK Activity Calculation

1. Based on Sample Mass:

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

• Formula:
  PGK (nmol/min/g fresh weight) = [ΔA ÷ (ε × d) × V2 × 10⁹] ÷ (W × V1 ÷ V) ÷ T = 156.8 × ΔA ÷ W

2. Based on Sample Protein Concentration:

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

• Formula:
  PGK (nmol/min/mg prot) = [ΔA ÷ (ε × d) × V2 × 10⁹] ÷ (V1 × Cpr) ÷ T = 156.8 × ΔA ÷ Cpr

3. Based on Bacterial/Cell Count:

• Unit Definition: One unit of enzyme activity is defined as the consumption of 1 nmol NADH per minute per 10⁴ cells.

• Formula:
  PGK (nmol/min/10⁴ cell) = [ΔA ÷ (ε × d) × V2 × 10⁹] ÷ (500 × V1 ÷ V) ÷ T = 0.314 × ΔA

Parameter Description:

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

• d: Cuvette light path, 1 cm

• V: Volume of Extraction Buffer added, 1 mL

• V1: Volume of sample supernatant added, 0.08 mL

• V2: Total reaction volume, 0.78 mL = 7.8 × 10⁻⁴ L

• T: Reaction time, 10 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

Protected from light,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.