BioReagent 级 ，适用于对基线干扰要求严格的色谱和分析工作流程。
避光,-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。
磷酸烯醇式丙酮酸羧激酶（Phosphoenol Pyruvate Carboxykinase, PEPCK, EC4.1.1.32）广泛存在于动物、植物、微生物和细胞中，催化草酰乙酸转化为磷酸烯醇式丙酮酸，是调节糖异生途径的关键酶。
测定原理： PEPCK催化草酰乙酸生成磷酸烯醇式丙酮酸和CO²
，丙酮酸激酶和乳酸脱氢酶进一步依次催化NADH 氧化生成NAD+，在340nm下测定NADH下降速率，即可反映PEPCK活性。
| Component | 50T | Storage |
| 酸性提取液 | 60 mL | 2-8℃ |
| 试剂一 | 45 mL | 2-8℃ |
| 试剂二 | 41 μL | 2-8℃ |
| 试剂三 | 1EA | -20℃ |
| 试剂四 | 1EA | -20℃ |
自备仪器和用品
紫外分光光度计、台式离心机、可调式移液器、1ml石英比色皿、研钵、冰和蒸馏水。
样本的前处理：
1、细菌或培养细胞：先收集细菌或细胞到离心管内，离心后弃上清；按照细菌或细胞数量（10⁴个）：提 取液体积（μl）为500~1000：1的比例（建议500万细菌或细胞加入1μl提取液），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）；8000g 4℃离心10min，取上清，置冰上待测。
2、组织：按照组织质量（g）：提取液体积(μl)为1：5~10的比例（建议称取约0.1g组织，加入1μl提取液），进行冰浴匀浆。8000g 4℃离心10min，取上清，置冰上待测。
3、血清（浆）样品：直接检测。
测定步骤：
1、分光光度计预热30min以上，调节波长至340nm，蒸馏水调零。
2、工作液的配制：临用前将试剂二和试剂三转移到试剂一中混合溶解待用；用不完的试剂分装后-20℃保存，禁止反复冻融。
3、试剂四的配制：临用前加入2.5mL蒸馏水充分溶解待用；用不完的试剂分装后-20℃保存，禁止反复冻融。
4、将工作液和试剂四置于37℃(哺乳动物)或25℃(其它物种)预热5分钟。
5、在1mL石英比色皿中加入50μl样本、50μl试剂四和900μl工作液，立即混匀，记录340nm处初始吸光值A1和 1min后的吸光值A2，计算ΔA=A1-A2。
注意：在该试剂盒中，若ΔA大于0.1，需将样本用提取液稀释适当倍数后测定，使ΔA小于0.1可提高检测灵敏度。计算公式中乘以相应稀释倍数。
PEPCK活性计算：
1、血清（浆）PEPCK活力计算
单位定义：每毫升血清（浆）每分钟消耗1nmol NADH定义为一个酶活力单位。
PEPCK（nmol/min/μl）＝[ΔA×V 反总÷（ε×d）× 10⁹ ]÷V样÷T=3215×ΔA
2、组织、细菌或细胞中PEPCK活力计算
2.1 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。
PEPCK（nmol/min/mg prot）＝[ΔA×V 反总÷（ε×d）× 10⁹ ]÷(V 样×Cpr) ÷T=3215×ΔA÷Cpr
2.2 按样本鲜重计算
单位定义：每g组织每分钟消耗1nmol NADH定义为一个酶活力单位。
PEPCK（nmol/min/g 鲜重）＝[ΔA×V反总÷（ε×d）× 10⁹ ]÷(W×V样÷V样总)÷T=3215×ΔA÷W
2.3 按细菌或细胞密度计算
单位定义：每1万个细胞每分钟消耗1nmol NADH定义为一个酶活力单位。
PEPCK（nmol/min/10⁹cell）＝[ΔA×V 反总÷（ε×d）× 10⁹ ]÷(500×V 样÷V 样总)÷T=6.43×ΔA
V反总：反应体系总体积，1×10⁻³L
ε：NADH摩尔消光系数，6.22×10 ³ L/mol/cm
d：比色皿光径，1cm
V样：加入样本体积，0.05μl
V样总：加入提取液体积，1μl
T：反应时间，1min
Cpr：样本蛋白质浓度，mg/μl
W：样本质量，g
500：细菌或细胞总数，500万
注意事项
正式测定前务必取2-3个预期差异较大的样本做预测定。
Phosphoenolpyruvate Carboxykinase (PEPCK, EC 4.1.1.32) is widely present in animals, plants, microorganisms, and cells. It catalyzes the conversion of oxaloacetate to phosphoenolpyruvate and is a key regulatory enzyme in the gluconeogenesis pathway.
Assay Principle
PEPCK catalyzes the conversion of Oxaloacetate to Phosphoenolpyruvate and CO₂. Pyruvate Kinase and Lactate Dehydrogenase subsequently catalyze the sequential oxidation of NADH to NAD⁺. The rate of decrease in NADH absorbance at 340 nm is measured, which reflects PEPCK activity.
| Component | 50T | Storage |
| Acidic Extraction Buffer | 60 mL | 2-8℃ |
| Reagent 1 | 45 mL | 2-8℃ |
| Reagent 2 | 41 μL | 2-8℃ |
| Reagent 3 | 1EA | -20℃ |
| Reagent 4 | 1EA | -20℃ |
Required Materials and Equipment (Not Provided)
UV spectrophotometer, benchtop centrifuge, adjustable pipettes, 1 ml quartz cuvette, mortar and pestle, ice, and distilled water.
Sample Preparation:
*Note: The provided sample-to-buffer ratios (1:1, w/v or based on cell count) using microliters (μl) are highly unusual and likely a typo in the original text. Standard protocols use milliliters (ml). The calculations also imply ml. The following protocol assumes the intended volumes are in milliliters (ml).*
Bacteria or Cultured Cells:
Collect cells by centrifugation and discard the supernatant.
Add Acidic Extraction Buffer at a ratio of 1 ml per 5-10 million cells (e.g., 1 ml for 5 million cells).
Sonicate on ice (20% power or 200W, pulse 3s on/10s off, repeat 30 times).
Centrifuge at 8000 g, 4°C for 10 min. Collect the supernatant and keep it on ice for assay.
Tissues:
Homogenize tissue on ice in Acidic Extraction Buffer at a ratio of 1:5-10 (w/v) (e.g., 0.1 g tissue in 1 ml buffer).
Centrifuge at 8000 g, 4°C for 10 min. Collect the supernatant and keep it on ice for assay.
Serum (or Plasma) Samples:
Assay directly.
Assay Procedure:
Preheat the spectrophotometer for at least 30 minutes. Set the wavelength to 340 nm. Zero the instrument with distilled water.
Preparation of Working Solution: Just before use, transfer and dissolve Reagent 2 and Reagent 3 into Reagent 1. Mix well. Aliquot and store any unused portions at -20°C. Avoid repeated freeze-thaw cycles.
Preparation of Reagent 4: Just before use, dissolve the contents of the vial in 2.5 mL of distilled water. Mix well. Aliquot and store any unused portions at -20°C. Avoid repeated freeze-thaw cycles.
Pre-warm the Working Solution and dissolved Reagent 4 at 37°C (for mammalian samples) or 25°C (for other species) for 5 minutes.
In a 1 ml quartz cuvette, add:
50 μl sample
50 μl dissolved Reagent 4
900 μl pre-warmed Working Solution
Mix immediately and record the initial absorbance (A₁) at 340 nm. Record the absorbance again (A₂) after exactly 1 minute. Calculate ΔA = A₁ - A₂.
Note: For this kit, if ΔA is greater than 0.1, dilute the sample with Acidic Extraction Buffer by an appropriate factor (account for this dilution factor 'n' in the calculations) so that ΔA is less than 0.1 to improve detection sensitivity.
PEPCK Activity Calculation:
General Parameters for 1 ml Cuvette (d = 1.0 cm):
Vₜₒₜₐₗ (Total reaction volume) = 0.001 L (1000 μL)
ε (NADH molar extinction coefficient) = 6220 L/mol/cm
d (Cuvette light path) = 1.0 cm
Vₛₐₘₚₗₑ (Sample volume in reaction) = 0.05 mL (50 μL) [Note: Corrected from 0.05μL, which is implausible]
T (Reaction time) = 1 min
Vₛₐₘₚₗₑₜₒₜₐₗ (Total extract volume) = 1 mL (for tissues/cells) [Note: Corrected from 1μL]
Cpr (Sample protein concentration, mg/mL) [Note: Corrected from mg/μL]
W (Sample mass, g)
500 (Cell/Bacteria count in millions for example calculation: 5 million)
1. For Serum (Plasma):
Definition: One unit of activity is defined as the amount of enzyme that consumes 1 nmol of NADH per minute per ml of serum.
Calculation:
PEPCK Activity (nmol/min/ml) = [ΔA × Vₜₒₜₐₗ ÷ (ε × d) × 10⁹] ÷ Vₛₐₘₚₗₑ ÷ T
Simplified Formula: PEPCK (nmol/min/ml) = 3215 × ΔA
2. For Tissues, Bacteria, or Cells:
Based on Sample Protein Concentration:
Definition: One unit of activity is defined as the amount of enzyme that consumes 1 nmol of NADH per minute per mg of protein.
Calculation:
PEPCK Activity (nmol/min/mg prot) = [ΔA × Vₜₒₜₐₗ ÷ (ε × d) × 10⁹] ÷ (Vₛₐₘₚₗₑ × Cpr) ÷ T
Simplified Formula: PEPCK (nmol/min/mg prot) = 3215 × ΔA ÷ Cpr
Based on Sample Fresh Weight:
Definition: One unit of activity is defined as the amount of enzyme that consumes 1 nmol of NADH per minute per gram of fresh tissue.
Calculation:
PEPCK Activity (nmol/min/g fresh weight) = [ΔA × Vₜₒₜₐₗ ÷ (ε × d) × 10⁹] ÷ (W × Vₛₐₘₚₗₑ / Vₛₐₘₚₗₑₜₒₜₐₗ) ÷ T
Simplified Formula: PEPCK (nmol/min/g fresh weight) = 3215 × ΔA ÷ W
Based on Bacterial or Cell Density:
Definition: One unit of activity is defined as the amount of enzyme that consumes 1 nmol of NADH per minute per 10⁴ cells.
Calculation (example for 5 million cells in 1 ml extract):
PEPCK Activity (nmol/min/10⁴ cell) = [ΔA × Vₜₒₜₐₗ ÷ (ε × d) × 10⁹] ÷ (500 × Vₛₐₘₚₗₑ / Vₛₐₘₚₗₑₜₒₜₐₗ) ÷ T
Simplified Formula: PEPCK (nmol/min/10⁴ cell) = 6.43 × ΔA
Precautions
Before formal assay, it is essential to perform a pilot test with 2-3 samples expected to have significant differences in activity.
危险品化学品经营许可证（带存储）