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6-P-G (Glucose-6-phosphate，葡萄糖-6-磷酸，又称6-磷酸葡萄糖)，是糖酵解和磷酸戊糖途径的中间产物，广泛存在于动植物体和微生物中。在糖酵解的第一步反应中，葡萄糖被己糖激酶催化生成葡萄糖-6-磷酸，然后通过磷酸葡萄糖异构酶的催化形成果糖-6-磷酸，以继续糖酵解的其它步骤；而在戊糖磷酸途径中，葡萄糖-6-磷酸是其第一个底物，该过程也是生成NADPH的主要途径。此外，葡萄糖-6-磷酸也能转化形成糖原或淀粉而被储存起来。
测定原理：
6-磷酸葡萄糖脱氢酶可催化6-P-G和NADP⁺生成6磷酸葡萄糖酸和NADPH，NADPH在PMS的作用下使WST-8显橙黄色，吸光度450nm处6-P-G的含量与吸光值成正比。
试剂配制:
试剂2：每瓶临用前加入4mL蒸馏水，充分溶解待用，用不完可分装后-20℃保存1周，禁止反复冻融。
试剂3：临用前加入3mL蒸馏水，充分溶解待用，用不完可分装后-20℃保存1周，禁止反复冻融。
标准品：加入1mL的蒸馏水，充分混匀，即为10μmol/mL的6-P-G标准母液，用于制备标准曲线，母液可于-20℃保存4周。(选做)
需自备仪器和用品：
分光光度计、台式离心机、可调式移液器、电子天平、1mL玻璃比色皿、研钵、冰、蒸馏水。
样本处理：
1、细菌或培养细胞：收集细菌或细胞到离心管内，离心后弃上清；按细菌或细胞数量(104个)：提取液体积(mL)为500~1000：1的比例(建议500万细菌或细胞加入1mL提取液)，超声波破碎(冰浴，功率20％或200W，超声3s，间隔10s，重复30次)， 8000g，4℃离心10min，取上清待测。
2、组织：按照组织质量(g)：提取液体积(mL)为1：5~10的比例(建议称取约0.1g组织，加入1mL提取液)，进行冰浴匀浆，8000g，4℃离心10min，取上清待测。
3、血清(浆)等液体样品：直接测定。
测定步骤：
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计算公式：
标准条件下测定的回归方程为y=8.3633x-0.0018，R²=0.9988，x为6-P-G浓度(μmol/mL)，y为吸光值。
1、液体样本中6-P-G含量的计算:
2、组织、细菌或细胞中6-P-G含量的计算:
(1)按样本蛋白浓度计算：
6-P-G含量(μmol/mg prot)＝(ΔA+0.0018)÷8.3633×V样÷V样÷Cpr=0.12×(ΔA+0.0018)÷Cpr
蛋白浓度需额外测定，建议使用本公司生产的BCA蛋白浓度测定试剂盒。
(2)按样本鲜重计算：
6-P-G含量(μmol /g 鲜重)＝(ΔA+0.0018)÷8.3633×V样÷(W×V样÷V样总)=0.12×(ΔA+0.0018)÷W
(3)按细菌或细胞密度计算：
6-P-G含量(μmol /104 cell)＝(ΔA+0.0018)÷8.3633×V样总÷500=0.12×(ΔA+0.0018)÷500
注意事项：
1. ΔA线性范围为0.04-2.1，可测定6-P-G浓度范围0.005-0.25μmol/mL；如果样本ΔA大于2.1，建议稀释待测样本后重新测定(计算公式中乘以相应稀释倍数)，如果ΔA小于0.04，建议适当增加样本量。
实验实例：
1. 分别取0.1311g牛肝和0.0983g牛心脏作为检测样本，按步骤提取,，用提取液分别稀释2倍后测定吸光值，结果如下：
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常见问题及解决方案：
1.样品读数重复性差
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2.样品和标准品读数偏低/偏高
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6-P-G (Glucose-6-phosphate, abbreviated as G6P) is an intermediate of glycolysis and the pentose phosphate pathway, widely distributed in animals, plants and microorganisms.
In the first step of glycolysis, glucose is catalyzed by hexokinase to produce glucose-6-phosphate, which is then converted into fructose-6-phosphate under the catalysis of phosphoglucose isomerase to proceed with subsequent glycolytic reactions. In the pentose phosphate pathway, glucose-6-phosphate acts as the initial substrate, and this pathway serves as the major source of NADPH generation. In addition, glucose-6-phosphate can be converted into glycogen or starch for energy storage.
Assay Principle
Glucose-6-phosphate dehydrogenase catalyzes the reaction between 6-P-G and NADP⁺ to produce 6-phosphogluconate and NADPH. In the presence of PMS, NADPH reduces WST-8 to form an orange-yellow product. The absorbance measured at 450 nm is linearly proportional to the concentration of 6-P-G.
Reagent Preparation
Reagent 2: Add 4 mL of distilled water to each vial before use and dissolve completely for immediate assay. Remaining solution can be aliquoted and stored at -20 °C for up to 1 week; repeated freeze-thaw cycles are prohibited.
Reagent 3: Add 3 mL of distilled water before use and dissolve completely for immediate assay. Remaining solution can be aliquoted and stored at -20 °C for up to 1 week; repeated freeze-thaw cycles are prohibited.
Standard Reference: Add 1 mL of distilled water and mix thoroughly to prepare the 10 μmol/mL 6-P-G standard stock solution for standard curve construction. The stock solution can be stored at -20 °C for 4 weeks. (Optional step)
Required Self-Prepared Equipment & Consumables
Spectrophotometer, tabletop centrifuge, adjustable pipettors, analytical balance, 1 mL glass cuvettes, mortar, ice, distilled water
Sample Pretreatment
Bacteria or cultured cells:Collect bacteria/cells into centrifuge tubes and discard the supernatant after centrifugation. Add extraction buffer at a ratio of 500–1000:1 (10⁴ cells : mL extraction buffer; recommended: 1 mL extraction buffer per 5 million bacteria/cells). Lyse cells by sonication on ice (power: 20% or 200 W; sonicate for 3 s, pause for 10 s, repeat 30 cycles). Centrifuge at 8000 g, 4 °C for 10 min, and retain the supernatant for testing.
Tissue samples:Add extraction buffer at a mass-to-volume ratio of 1 g tissue : 5–10 mL extraction buffer (recommended: 1 mL extraction buffer for ~0.1 g tissue). Homogenize on ice, then centrifuge at 8000 g, 4 °C for 10 min, and retain the supernatant for testing.
Liquid samples such as serum/plasma:Measure directly without pretreatment.
Assay Procedure
Volumes shown in microliters (µL)
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Calculation Formula
The regression equation obtained under standard assay conditions is y=8.3633x−0.0018, with R²=0.9988.
Where:
x = concentration of 6-P-G (μmol/mL)
y = absorbance value
Calculation Formulas
1. Calculation of 6-P-G Content in Liquid Samples
6-P-G content (μmol/mL) = (ΔA + 0.0018) ÷ 8.3633 × Vsample ÷ Vsample = 0.12 × (ΔA + 0.0018)
2. Calculation of 6-P-G Content in Tissues, Bacteria or Cultured Cells
(1) Calculation based on sample protein concentration
6-P-G content (μmol/mg prot) = (ΔA + 0.0018) ÷ 8.3633 × Vsample ÷ Vsample ÷ Cpr = 0.12 × (ΔA + 0.0018) ÷ Cpr
The protein concentration needs to be measured separately. The BCA Protein Quantification Kit manufactured by our company is recommended.
(2) Calculation based on fresh weight of samples
6-P-G content (μmol/g fresh weight) = (ΔA + 0.0018) ÷ 8.3633 × Vsample ÷ (W × Vsample ÷ Vtotal extract) = 0.12 × (ΔA + 0.0018) ÷ W
(3) Calculation based on bacterial or cell density
Precautions
The linear range of ΔA is 0.04–2.1, corresponding to a detectable 6-P-G concentration range of 0.005–0.25 μmol/mL.
If the ΔA value of the sample exceeds 2.1, dilute the sample and re-test (multiply the final result by the corresponding dilution factor). If ΔA is lower than 0.04, appropriately increase the sample loading volume.
Aqueous solution of Reagent 3 is light purplish red under normal conditions. Discard the reagent immediately and discontinue use if the solution becomes colorless, as it has lost activity.
Experimental Examples
1.Take 0.1311 g bovine liver and 0.0983 g bovine heart as test samples respectively, extract samples in accordance with the procedures, dilute the extracts 2-fold with extraction buffer, then measure absorbance values. The results are shown below:
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2. Undiluted chicken serum was tested directly. A<sub>assay</sub> = 0.343, A<sub>control</sub> = 0.293, ΔA = 0.05. The 6-P-G content in chicken serum is 0.006 μmol/mL.
Frequently Encountered Problems and Troubleshooting
1. Poor repeatability of sample absorbance readings
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2. Abnormally low or high absorbance readings for samples and standards
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| G1522113 | 组件 | 物理外观 | 50T | 储存 |
| G1522113A | 提取液 | 液体 | 60mL | 2-8 ℃ |
| G1522113B | 试剂1 | 液体 | 30mL | 2-8 ℃ |
| G1522113C | 试剂2 | 粉末 | 2瓶 | -20℃. Store in the dark. |
| G1522113D | 试剂3 | 粉末 | 1瓶 | -20℃. Store in the dark. |
| G1522113E | 标准品 | 粉末 | 1瓶 | -20℃. Store in the dark. |
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| 批号(Lot Number) | 证书类型 | 货号 |
|---|---|---|
 I2624328 |
分析证书 | G1522113 |
| G2629155 |
分析证书 | G1522113 |
| ZJ26F0737659 |
分析证书 | G1522113 |
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