BioReagent 级 ，适用于对基线干扰要求严格的色谱和分析工作流程。
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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。
3-磷酸甘油酸激酶（3-Phosphoglycerate Kinase, PGK）是糖酵解的关键酶，广泛存在于动植物和微生物体内，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(PGK)的活性大小。
| Component | 100T | Storage |
| 提取液 | 100 mL | 2-8℃. Store in the dark. |
| 试剂一 | 1EA | -20℃. Store in the dark. |
| 试剂二 | 3EA | 2-8℃ |
| 试剂三 | 1EA | -20℃ |
| 试剂四 | 15 mL | 2-8℃ |
| 试剂五 | 1EA | -20℃ |
试剂一（粉剂1瓶）：
1、临用前8000g 4°C 离心 2min使粉剂落入管底（可手动甩一甩）；
2、加入1.1mL蒸馏水溶解备用；
3、溶解好的试剂可-20℃分装冻存。
试剂二（粉剂3支）：
1、临用前8000g 4°C 离心 2min使粉剂落入管底（可手动甩一甩）；
2、加入0.4mL蒸馏水溶解备用；
3、溶解好的试剂可-20℃分装冻存（溶解好的一个月内用完）。
试剂三（液体1支）：
1、临用前8000g 4°C离心 2min使试剂落入管底（可手动甩一甩）；
2、加入1.1 mL蒸馏水溶解备用，溶解好的试剂可-20℃分装冻存。
试剂五（粉剂1支）：
1、临用前8000g 4°C离心 2min使试剂落入管底（可手动甩一甩）；
2、加入1.1 mL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、蒸馏水（去离子水、超纯水均可）。
样本提取
1、组织样本：称取约0.1g组织样本，加入1mL提取液，冰浴匀浆后于4℃，12000rpm离心5min，取上清液体待测。
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、细菌/细胞样本：先收集细菌或细胞到离心管内，离心后弃上清；取约500万细菌或细胞加入1mL提取液，超声波破碎细菌或细胞（冰浴，功率200W，超声3s，间隔10s，重复30次）；12000rpm 4℃离心10min，取上清，置冰上待测。
注：若增加样本量，可按照细菌/细胞数量（10⁴）：提取液（mL）为500~1000：1的比例进行提取。
测定步骤
1、酶标仪预热30min，调节波长至340nm，设定温度25℃。
2、所有试剂解冻至室温（25℃）。
3、在96孔板中依次加入：
| 试剂（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。
PGK活性计算
1、按照样本质量计算
酶活定义：每g组织每分钟消耗1nmol NADH定义为一个酶活力单位。
GPK (nmol/min/g鲜重)=[ΔA÷(ε×d)×V2× 10⁹ ]÷(W×V1÷V)÷T=321.6×ΔA÷W
2、按样本蛋白浓度计算
单位定义：每mg组织蛋白每分钟消耗1nmol NADH定义为一个酶活力单位。
GPK (nmol/min/mg prot)=[ΔA÷(ε×d)×V2× 10⁹ ]÷(V1×Cpr)÷T=321.6×ΔA÷Cpr
3、按细菌/细胞数量计算
单位定义：每10⁴个细胞每分钟消耗1nmol NADH定义为一个酶活力单位。
GPK (nmol/min/10⁴ cell)=[ΔA÷(ε×d)×V2× 10⁹ ]÷(500×V1÷V)÷T=0.64×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；
500：细胞数量，万；
Cpr：上清液蛋白质浓度，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。
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 | 100T | Storage |
| Extraction Buffer | 100 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 | 15 mL | 2-8℃ |
| Reagent 5 | 1EA | -20℃ |
Reagent Preparation:
Reagent 1 (Powder, 1 vial):
Before use, centrifuge at 8000 g, 4°C for 2 min to collect the powder at the bottom (tap manually if needed).
Add 1.1 mL of distilled water to dissolve.
The dissolved reagent can be aliquoted and stored at -20°C.
Reagent 2 (Powder, 3 vials):
Before use, centrifuge at 8000 g, 4°C for 2 min to collect the powder at the bottom (tap manually if needed).
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):
Before use, centrifuge at 8000 g, 4°C for 2 min to collect the liquid at the bottom (tap manually if needed).
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):
Before use, centrifuge at 8000 g, 4°C for 2 min to collect the powder at the bottom (tap manually if needed).
Add 1.1 mL of distilled water to dissolve.
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), 96-well plate, centrifuge tubes, microplate reader, 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 microplate reader for 30 minutes. Set the wavelength to 340 nm and the temperature to 25°C.
2. Thaw all reagents to room temperature (25°C).
3. In a well of the 96-well plate, add sequentially:
| Reagent (μL) | Test Tube |
| 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.
4. Add
| Reagent (μL) | Test Tube |
| Reagent 5 | 10 |
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 (T) must be substituted into the calculation formula. Alternatively, increase the sample volume appropriately (e.g., to 40 μL, with a corresponding decrease in Reagent 4 volume). The modified sample volume (V1) 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 (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.
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 10 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 = 321.6 × 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 = 321.6 × 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 = 0.64 × ΔA
Parameter Description:
ε: NADH molar extinction coefficient, 6.22 × 10³ L/mol/cm
d: Light path of the 96-well plate, 0.5 cm
V: Volume of Extraction Buffer added, 1 mL
V1: Volume of sample supernatant added, 0.02 mL
V2: Total reaction volume, 0.2 mL = 2.0 × 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.
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