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。
葡萄糖激酶（GK, EC2.7.1.2）是己糖激酶家族中的一员，主要存在于成熟的肝细胞和胰岛细胞中。 在正常的生理条件下，GK的主要作用是监控血中葡萄糖水平。
葡萄糖激酶(GK)磷酸化葡萄糖并产生6-磷酸葡萄糖，该产物进一步与6-磷酸葡萄糖脱氢酶和 NADP+偶联，在340nm测NADPH光吸收增加量，进而计算出该酶活性大小。
| Component | 50T | Storage |
| 提取液 | 60 mL | 2-8℃ |
| 试剂一 | 40 mL | 2-8℃ |
| 试剂二 | 1EA | -20℃ |
| 试剂三 | 1EA | 2-8℃ |
试剂二（粉剂1瓶）：
1、开盖前注意使粉体落入底部（可手动甩一甩）；
2、加2.2mL的蒸馏水溶解备用；
3、保存周期与试剂盒有效期相同。
试剂三（粉剂1瓶）：
1、开盖前注意使粉体落入底部（可手动甩一甩）；
2、加入36mL的试剂一溶解备用；
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）、 1ml 石英比色皿、离心管、紫外分光光度计、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，加入1mL提取液，进行冰浴匀浆。12000rpm，4℃离心10min，取上清，置冰上待测。
注：若增加样本量，可以按照组织质量（g）：提取液体积(mL)为1：5~10的比例提取。
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500万细菌或细胞加入1mL提取液；冰浴超声波破碎细菌或细胞（冰浴，功率20％或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，可按照细菌或细胞数量（10⁴个）：提取液体积（mL）为500~1000：1的比例进行提取。
3、液体样本：直接检测。若浑浊，离心后取上清检测。
测定步骤
1、紫外分光光度计预热30min以上，调节波长至340nm，蒸馏水调零。
2、配置好的试剂二和三在25℃预热5min至室温。
3、在1mL石英比色皿（光径1cm）中依次加入：
| 试剂（μL） | 测定管 |
| 样本 | 80 |
| 试剂二 | 40 |
| 试剂三 | 680 |
混匀，1min时于340nm处读取吸光值A1，21min（即 20min 后）读取A2，ΔA=A2-A1。
注：
1、若ΔA的值在零附近，可以适当延长反应时间到30min或更长读取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，则改变后的加样体积V1需代入计算公式重新计算。
2、若上升趋势不稳定，可以每隔10s读取一次吸光值，选取一段线性上升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。
GK活性计算
1、按样本蛋白浓度计算
单位定义：每毫克组织蛋白每分钟生成1nmol NADPH定义为一个酶活力单位。
GK (nmol/min/mg prot)=[ΔA÷(ε×d)×V2× 10⁹]÷(V1×Cpr) ÷T=80.4×ΔA÷Cpr
2、按样本鲜重计算
单位定义：每g组织每分钟生成1nmol NADPH定义为一个酶活力单位。
GK (nmol/min/g 鲜重)=[ΔA÷(ε×d)×V2× 10⁹]÷(W×V1÷V) ÷T=80.4×ΔA÷W
3、按细菌或细胞密度计算
单位定义：每1万个细菌或细胞每分钟生成1nmol NADPH定义为一个酶活力单位。
GK (nmol/min/10⁴ cell)=[ΔA÷(ε×d)×V2× 10⁹]÷(500×V1÷V)÷T=0.16×ΔA
4、按液体体积计算
单位定义：每毫升液体在每分钟生成1nmol NADPH定义为一个酶活力单位。
GK (nmol/min/mL)=[ΔA×V 反总÷(ε×d)× 10⁹]÷V1÷T=80.4×ΔA
参数说明
ε：NADH摩尔消光系数，6.22×10³ L/mol/cm；
d：1mL石英比色皿光径，1cm；
V：加入提取液体积，1mL；
V1：加入样本体积，0.08mL；
V2：反应体系总体积，0.8mL=8×10⁻⁴ L；
T：反应时间，20min；
W：样本质量，g；
500：细菌或细胞数量，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。
Glucokinase (GK, EC 2.7.1.2) is a member of the hexokinase family, primarily found in mature hepatocytes and pancreatic islet cells. Under normal physiological conditions, the main role of GK is to monitor blood glucose levels.
Glucokinase (GK) phosphorylates glucose to produce glucose-6-phosphate. This product is further coupled with glucose-6-phosphate dehydrogenase and NADP+, and the increase in NADPH absorbance is measured at 340 nm, thereby calculating the enzyme activity.
| Component | 50T | Storage |
| Extraction Buffer | 60 mL | 2-8℃ |
| Reagent 1 | 40 mL | 2-8℃ |
| Reagent 2 | 1EA | -20℃ |
| Reagent 3 | 1EA | 2-8℃ |
Reagent Preparation
Reagent 2 (Powder, 1 vial):
Before opening, ensure the powder is at the bottom of the vial (tap manually if needed).
Add 2.2 mL of distilled water to dissolve.
The storage period is the same as the kit's expiry date.
Reagent 3 (Powder, 1 vial):
Before opening, ensure the powder is at the bottom of the vial (tap manually if needed).
Add 36 mL of Reagent 1 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), 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, and homogenize on ice. 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 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 5 million bacteria or cells, add 1 mL of Extraction Buffer, and disrupt using ultrasound on ice (power 20% or 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.
3. Liquid Samples: Detect directly. If the sample is turbid, centrifuge and use the supernatant for detection.
Assay Procedure
1. Preheat the UV spectrophotometer for at least 30 minutes. Set the wavelength to 340 nm. Zero the instrument with distilled water.
2. Pre-warm the prepared Reagent 2 and Reagent 3 at 25°C for 5 minutes to reach room temperature.
3. In a 1 mL quartz cuvette (1 cm light path), add sequentially:
| Reagent (μL) | Test Cuvette |
| Sample | 80 |
| Reagent 2 | 40 |
| Reagent 3 | 680 |
4. Mix well. Read the absorbance at 340 nm at 1 minute (A1) and then again at 21 minutes (i.e., after 20 minutes) (A2). Calculate ΔA = A2 - A1.
Notes:
1. If ΔA is close to zero, the reaction time can be appropriately extended to 30 minutes or longer before reading A2. The modified reaction time (T) must be substituted into the calculation formula. Alternatively, increase the sample volume appropriately. The modified sample volume (V1) must be substituted into the calculation formula.
2. If the increasing trend is unstable, read the absorbance every 10 seconds and select a linear increasing period for calculation. The corresponding ΔA value should be substituted into the calculation formula.
GK Activity Calculation
1. Based on Sample Protein Concentration:
Unit Definition: One unit of enzyme activity is defined as the generation of 1 nmol NADPH per minute per mg of protein.
Formula:
GK (nmol/min/mg prot) = [ΔA ÷ (ε × d) × V2 × 10⁹] ÷ (V1 × Cpr) ÷ T = 80.4 × ΔA ÷ Cpr
2. Based on Sample Fresh Weight:
Unit Definition: One unit of enzyme activity is defined as the generation of 1 nmol NADPH per minute per gram of fresh tissue.
Formula:
GK (nmol/min/g fresh weight) = [ΔA ÷ (ε × d) × V2 × 10⁹] ÷ (W × V1 ÷ V) ÷ T = 80.4 × ΔA ÷ W
3. Based on Bacterial/Cell Density:
Unit Definition: One unit of enzyme activity is defined as the generation of 1 nmol NADPH per minute per 10⁴ bacteria/cells.
Formula:
GK (nmol/min/10⁴ cell) = [ΔA ÷ (ε × d) × V2 × 10⁹] ÷ (500 × V1 ÷ V) ÷ T = 0.16 × ΔA
4. Based on Liquid Volume:
Unit Definition: One unit of enzyme activity is defined as the generation of 1 nmol NADPH per minute per mL of liquid.
Formula:
GK (nmol/min/mL) = [ΔA × V2 ÷ (ε × d) × 10⁹] ÷ V1 ÷ T = 80.4 × ΔA
Parameter Description:
ε: NADPH molar extinction coefficient, 6.22 × 10³ L/mol/cm
d: Light path of the 1 mL quartz cuvette, 1 cm
V: Volume of Extraction Buffer added, 1 mL
V1: Volume of sample supernatant added, 0.08 mL
V2: Total reaction volume, 0.8 mL = 8 × 10⁻⁴ L
T: Reaction time, 20 min
W: Sample mass, g
500: Bacteria or 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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