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

Cat. No.: C1505414

GRADE & PURITY BioReagent

Descripción general

  Chloroplast 3-phosphoglycerate kinase (PGK) is a key enzyme in the Calvin cycle.

  Detection Principle: Chloroplast 3-phosphoglycerate kinase catalyzes the reaction of 3-phosphoglyceric acid and ATP to produce 1,3-bisphosphoglyceric acid. The latter, under the action of glyceraldehyde-3-phosphate dehydrogenase and NADH, produces glyceraldehyde-3-phosphate and NAD⁺. The enzyme activity of 3-phosphoglycerate kinase is determined by measuring the decrease in NADH.

Component	96T	Storage
Extraction Buffer 1	100 mL	2-8℃
Extraction Buffer 2	100 mL	2-8℃
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 (can be flicked manually).

  2. Add 1.1 mL of distilled water to dissolve. Use after preparation.

  3. The prepared solution can be stored for the duration of the kit's validity period.

• Reagent 2 (Powder, 3 vials):

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

  2. Add 0.4 mL of distilled water per vial to dissolve. Use after preparation.

  3. Unused dissolved reagent can be aliquoted and stored at -20°C. Avoid repeated freeze-thaw cycles. Use within 3 days.

• Reagent 3 (Powder, 1 vial):

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

  2. Add 1.1 mL of distilled water to dissolve. Use after preparation.

  3. The prepared solution can be stored for the duration of the kit's validity period.

• Reagent 5 (Powder, 1 vial):

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

  2. Add 1.1 mL of distilled water to dissolve. Use after preparation.

  3. The prepared solution can be stored for the duration of the kit's validity period.

User-Prepared Instruments and Materials
Mortar (Homogenizer), Ice box (Ice maker), Benchtop centrifuge, Adjustable micropipettes, Water bath (Oven, Incubator, Metal bath), 96-well plate, Centrifuge tubes, Microplate reader, Vortex mixer/shaker, Distilled water (Deionized water or Ultrapure water are acceptable).

Experimental Procedure

It is recommended to first perform a preliminary test using 1-3 samples with expected significant differences (e.g., different types or groups) to familiarize yourself with the procedure and to determine or adjust sample concentrations based on the preliminary results, preventing unnecessary waste of samples or reagents.

1. Sample Extraction (Chloroplast Isolation)
Weigh approximately 0.1 g of plant tissue sample. Add 1 mL of Extraction Buffer 1 and homogenize rapidly in an ice bath. Centrifuge at 1,600 rpm, 4°C for 5 minutes. Discard the pellet. Take the supernatant and centrifuge again at 5,000 rpm, 4°C for 15 minutes. Discard the supernatant and keep the pellet. Add 1 mL of Extraction Buffer 2 to the pellet. Vortex vigorously for 15 seconds. Place on ice (or in a refrigerator) and incubate at 4°C for 15 minutes. Centrifuge at 13,000 rpm, 4°C for 5 minutes. Collect the supernatant for assaying the chloroplast 3-phosphoglycerate kinase (PGK) enzyme activity.
Important: The entire chloroplast extraction process must be maintained at 4°C.
Note: If increasing the sample amount, maintain a tissue mass (g) to Extraction Buffer volume (mL) ratio between 1:5 and 1:10.

2. Assay Steps

2.1 Preheat the microplate reader for 30 minutes. Set the wavelength to 340 nm and the temperature to 25°C.
2.2 Thaw all reagents to room temperature (25°C).
2.3 Add reagents sequentially to a 96-well plate:

Reagent	Test Well (μL)
Sample	20
Reagent 1	10
Reagent 2	10
Reagent 3	10
Reagent 4	140

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

Reagent 5

10

Mix gently. Under room temperature (25°C) conditions, read the absorbance at 340 nm at 30 seconds (A₁) and then again after 10 minutes (A₂). Calculate ΔA = A₁ - A₂.
注：
Notes:
(1) If ΔA is close to zero, the reaction time can be appropriately extended to 20 minutes before reading A₂. If the reaction time is changed, the new time (T) must be substituted into the calculation formula. Alternatively, the sample volume can be increased (e.g., to 40 μL, with a corresponding decrease in Reagent 4 volume); the new sample volume (V₁) must then be substituted into the calculation formula.
(2) If the decrease trend is unstable, read the absorbance every 20 seconds and select a linearly decreasing time period for calculation. The corresponding A values for this period should be used to calculate ΔA and substituted into the formula.
(3) If the initial absorbance A₁ is too high (e.g., >2, as in dark green plant leaves with high pigment content), consider appropriately reducing the sample volume; the new sample volume (V₁) must be substituted into the calculation formula. Alternatively, add a small amount of activated carbon to the sample, mix, let stand for 5 minutes, then centrifuge at 12,000 rpm, 4°C for 10 minutes, and use the supernatant for assay.
(4) If ΔA is greater than 0.5, reduce the reaction time (e.g., to 5 minutes) or reduce the sample volume (e.g., to 10 μL). The changed reaction time (T) and/or sample volume (V₁) must be substituted into the calculation formula.

3. Calculation of Results

3.1 Based on Sample Mass
Unit Definition: One unit of enzyme activity is defined as the amount that consumes 1 nmol of NADH per minute per gram of tissue.

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

• Simplified Formula: chl PGK (nmol/min/g fresh weight) = 321.6 × ΔA ÷ W

3.2 Based on Sample Protein Concentration
Unit Definition: One unit of enzyme activity is defined as the amount that oxidizes 1 nmol of NADH per minute per mg of tissue protein.

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

• Simplified Formula: chl PGK (nmol/min/mg prot) = 321.6 × ΔA ÷ Cpr

Parameter Definitions:

• ε: Molar extinction coefficient of NADH (6.22 × 10³ L/mol/cm)

• d: Light path length for the 96-well plate (0.5 cm)

• V: Volume of Extraction Buffer added to the pellet (1 mL)

• V₁: Volume of sample added to the reaction (0.02 mL)

• V₂: Total volume of the reaction system (0.2 mL = 2.0 × 10⁻⁴ L)

• T: Reaction time (10 minutes)

• W: Sample weight (g)

• Cpr: Sample protein concentration (mg/mL); Aladdin's BCA Protein Quantification Kit (B665595) or Ready-to-Use BCA Protein Quantification Kit (R1491648) is recommended.

Precautions

  It is strongly recommended to first perform a preliminary test using 1-3 samples with expected significant differences (e.g., different types or groups) to familiarize yourself with the procedure. Based on the preliminary results, determine or adjust sample concentrations to prevent unnecessary waste of samples or reagents.

Almacenamiento y envío

Condiciones de almacenamiento de almacenamiento

Store at -20°C

Enviado en

Ice chest + Ice pads

Estabilidad y almacenamiento

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