BioReagent 级 ，适用于对基线干扰要求严格的色谱和分析工作流程。
-20°C储存。超低温运输 。请查阅批次 COA 获取详细规格。
SDS、COA、产品数据表及规格说明书均可下载。可通过批号查询获取批次 COA。
在色谱分析、有机合成和交叉偶联反应领域已被 0 篇同行评审文献引用。
叶绿体3-磷酸甘油酸激酶(PGK)是卡尔文循环中的关键酶。
检测原理：叶绿体3-磷酸甘油酸激酶催化3-磷酸甘油酸和ATP反应产生1,3 二磷酸甘油酸，后者在3-磷酸甘油醛脱氢酶和NADH作用下产生3-磷酸甘油醛和NAD+，通过测定NADH的下降量，进而得到3-磷酸甘油酸激酶的活性大小。
| Component | 96T | Storage |
| 提取液一 | 100 mL | 2-8℃ |
| 提取液二 | 100 mL | 2-8℃ |
| 试剂一 | 1EA | -20℃. Store in the dark. |
| 试剂二 | 3EA | 2-8℃ |
| 试剂三 | 1EA | -20℃ |
| 试剂四 | 35 mL | 2-8℃ |
| 试剂五 | 1EA | -20℃ |
试剂一（粉剂1支）：
1、开盖前注意使粉体落入底部（可手动甩一甩）；
2、加入1.1mL蒸馏水溶解备用；
3、保存周期与试剂盒有效期相同。
试剂二（粉剂3支）：
1、临用前8000g 4°C离心2min使试剂落入管底；
2、加入0.4mL蒸馏水溶解备用。用不完的试剂分装后-20℃保存，禁止反复冻融， 三天内用完。
试剂三（粉剂1支）：
1、临用前8000g 4°C离心2min使试剂落入管底；
2、加1.1mL蒸馏水溶解备用；
3、保存周期与试剂盒有效期相同。
试剂五（粉剂1支）：
1、临用前8000g 4°C离心2min使试剂落入管底；
2、加1.1mL蒸馏水溶解备用；
3、保存周期与试剂盒有效期相同。
自备仪器和用品
研钵（匀浆机）、冰盒（制冰机）、台式离心机、可调式移液枪、水浴锅（烘箱、培养箱、金属浴）、 96 孔板、离心管、酶标仪、震荡仪、蒸馏水（去离子水、超纯水均可）。
实验流程
建议先选取1-3个差异大的样本（例如不同类型或分组）进行预实验，熟悉操作流程，根据预实验结果确定或调整样本浓度，以防造成样本或试剂不必要的浪费！
1、样本提取
称取约0.1g植物组织样本，加入1mL提取液一，快速冰浴匀浆后于4℃，1600rpm离心5min，弃沉淀，取上清再4℃，5000rpm离心15min，弃上清留沉淀，向沉淀中加1mL提取液二，强力涡旋震 荡15s，置于冰上（或冰箱）在4℃孵育15min，4℃，13000rpm离心5min，取上清测定叶绿体中3-磷酸甘油酸激酶 (PGK) 的酶活性。提示：整个叶绿体的提取过程须保持4℃低温环境。
注：若增加样本量，可按照组织质量(g)：提取液体积(mL)为1：5~10的比例进行提取。
2、检测步骤
2.1 酶标仪预热30min，调节波长至340nm，设定温度25℃。
2.2 所有试剂解冻至室温（25℃）。
2.3 在96孔板中依次加入：
| 试剂组分 (μL) | 测定管 |
| 样本 | 20 |
| 试剂一 | 10 |
| 试剂二 | 10 |
| 试剂三 | 10 |
| 试剂四 | 140 |
混匀，室温（25℃）条件下，孵育10min。
| 试剂五 | 10 |
轻轻混匀，室温（25℃）条件下，30s时于340nm 处读取吸光值A1，10min后再读取A2，ΔA=A1-A2。
注：
(1) 若ΔA的值在零附近，可以适当延长反应时间到20min后读取A2，改变后的反应时间需代入计算公式重新计算。或适当加大样本量（如40μL，则试剂四相应减少），则改变后的加样体积需代入计算公式重新计算。
(2) 若下降趋势不稳定，可以每隔20s读取一次吸光值，选取一段线性下降的时间段来参与计算，相对应的A值也代入计算公式重新计算。
(3) 若起始值A1太大如超过2（如颜色较深的植物叶片，一般色素较高，则起始值相对会偏高），可以适当减少样本加样量，则改变后的加样体积需代入计算公式重新计算。或向待测样本中加少许活性炭混匀静置5min后12000rpm，4℃离心10min，上清液用于检测；
(4) 若ΔA的值大于0.5，则需减少反应时间（如减少至5min），或减少样本量（如10μL），则改变后的反应时间T和样本量V1需代入计算公式重新计算。
3、结果计算
3.1 按照样本质量计算
酶活定义：每克组织每分消耗1nmol的NADH定义为一个酶活力单位。
chl PGK (nmol/min/g鲜重)=[ΔA÷(ε×d)×V2×10⁹]÷(W×V1÷V)÷T=321.6×ΔA÷W
3.2 按样本蛋白浓度计算
单位定义：每毫克组织蛋白在每分钟内氧化1nmol的NADH定义为一个酶活力单位。
chl PGK (nmol/min/mg prot)=[ΔA÷(ε×d)×V2×10⁹]÷(V1×Cpr)÷T=321.6×ΔA÷Cpr
参数说明
ε：NADH摩尔消光系数，6.22×10³ L/mol/cm；
d：96孔板光径，0.5cm；
V：加入提取液体积，1mL；
V1：加入样本体积，0.02mL；
V2：反应体系总体积，0.2mL=2.0×10⁻⁴ L；
T：反应时间，10min；
W：样本质量，g；
Cpr：样本蛋白质浓度，mg/mL；推荐使用阿拉丁B665595 BCA蛋白定量试剂盒或R1491648 即用型BCA蛋白定量试剂盒。
注意事项
建议先选取1-3个差异大的样本（例如不同类型或分组）进行预实验，熟悉操作流程，根据预实验结果确定或调整样本浓度，以防造成样本或试剂不必要的浪费。
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):
Before opening, ensure the powder is at the bottom (can be flicked manually).
Add 1.1 mL of distilled water to dissolve. Use after preparation.
The prepared solution can be stored for the duration of the kit's validity period.
Reagent 2 (Powder, 3 vials):
Before use, centrifuge at 8000 g, 4°C for 2 minutes to collect the powder at the bottom.
Add 0.4 mL of distilled water per vial to dissolve. Use after preparation.
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):
Before use, centrifuge at 8000 g, 4°C for 2 minutes to collect the powder at the bottom.
Add 1.1 mL of distilled water to dissolve. Use after preparation.
The prepared solution can be stored for the duration of the kit's validity period.
Reagent 5 (Powder, 1 vial):
Before use, centrifuge at 8000 g, 4°C for 2 minutes to collect the powder at the bottom.
Add 1.1 mL of distilled water to dissolve. Use after preparation.
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.
危险品化学品经营许可证（带存储）