Solarbio Pyrophosphate: Fructose 6-phosphate-1 Phosphotransferase Assay Kit

屬性	細節
筆記	測試前取兩個或三個不同的樣本進行預測
檢測儀器	分光光度計
目錄號	BC3400
尺寸	50T/48S

成分:

萃取液: 55毫升× 1, 儲存於 4 ℃.

試劑 1: 40毫升× 1, 儲存於 4 °C and protected from light.

試劑 2: 粉末× 1, 儲存於 -20 °C and protected from light. Just before use, 添加 6 毫升蒸餾水以完全溶解. Unused reagents are stored at -20 °C 為 1 week after dispensing to avoid repeated freeze-thaw cycles.

試劑 3: powder × 1, 儲存於 -20 °C and protected from light. Just before use, 添加 6 毫升蒸餾水以完全溶解. Unused reagents are stored at -20 °C after dispensing. Prohibition of repeated freeze-thaw cycles.

試劑 4: 91µL × 2, 儲存於 4 °C and protected from light. Just before use, 添加 0.209 毫升蒸餾水以完全溶解. 存儲在 4 °C 為 1 week.

試劑 5: 粉末× 2, 儲存於 -20 °C and protected from light. Just before use, 添加 0.3 毫升蒸餾水以完全溶解. Unused reagents are stored at -20 °C 為 1 week after dispensing.

試劑 6: 60 µL × 1, 儲存於 4 °C and protected from light. Just before use, 添加 0.6 毫升蒸餾水以完全溶解. 存儲在 4 °C 為 1 week.

產品描述:

Pyrophosphate: Fructose-6-phosphate-1-phosphotransferase (PFP, EC2.7.1.90) is a cytosolic enzyme that is widely present in plant tissues and catalyzes the phosphorylation of fructose-6-phosphate like phosphofructokinase. As a result, the single PEP catalytic reaction is a reversible reaction, and pyrophosphate is used instead of ATP, which plays an important role in carbon metabolism of photosynthesis.

PFP catalyzes the conversion of fructose 6-phosphate to fructose 1,6-diphosphate, which is converted to dihydroxyacetone phosphate by the action of aldolase and triose phosphate isomerase, and then catalyzed by α-phosphate glycerol dehydrogenase and NADH to from Glycerol 3-diphosphate and NAD. The change in absorbance at 340 nm reflects the level of PFP activity.

所需的材料

Low temperature centrifuge, 分光光度計, water bath/constant temperature incubator, 研缽/均質器, 1 ML石英比色卷, 移液管, 冰和蒸餾水, EP管.

程式:

我. 樣本 萃取:

1. 1. 組織樣本:

根據組織的質量 (G): the volume of the extract solution (毫升) 是 1: 5 ～ 10. Suggested 0.1g of tissue with 1mL of extract solution. 完全在冰上磨, centrifugated at 20000g and 4℃ for 15 分分鐘. 上清液放在冰上進行測試.

2. 細菌或細胞:

根據細胞的數量 (104): the volume of the extract solution (毫升) 是 500 ～ 1000: 1. 推薦 5 million with 1mL of Extract Solution. 使用超聲處理分裂細菌或細胞 (功率300W, 工作時間3s, 間隔7秒, 總時間 3 分分鐘). 離心在, 20000g和4℃ 15 分分鐘. 上清液放在冰上進行測試.

3. Liquids: direct detection.

二. 決心 程式:

• 預熱分光光度計 30 分分鐘, 調整波長至 340 奈米, 用蒸餾器調零
• 加入如下清單的試劑:

試劑名稱 (微升)	試管 (時間)	空白管 (時間)
試劑 1	670	670
試劑 2	100	100
試劑 3	100	100
試劑 4	10	10
試劑 5	10	10
試劑 6	10	10
樣本	100	–
蒸餾水	–	100
徹底混合後, measure the initial value A1 at 340 nm and the absorbance A2 for 30 分鐘在 37 °C in a 1 ML石英比色卷, and record them as A1T, A1B, and A2T, A2B. Calculate ΔA = (A1T-A2T)- (A1B-A2B). 筆記: 試劑 1, 2, 3, 4, 5, 和 6 can also be formulated into working fluids according to the proportions of the operation table, which is now prepared for use; The blank tubes need only be made 1–2 times.

三、. Calculation of PFP 活動:

1 由微石英比色杯計算

• 通過蛋白質濃度計算:

單位定義: One unit of enzyme activity is defined as that 1 mg of tissue protein per minute consumes 1 nmol of NADH.

PFP activity (U/毫克蛋白質) =ΔA÷（ε×d）×VRT×109÷(VS×CPR) ÷T=53.59×ΔA÷Cpr

• 通過樣品重量計算

單位定義: One unit of enzyme activity is defined as that 1g of tissue per minute consumes 1 nmol of NADH.

PFP activity (U/g鮮重) =ΔA÷（ε×d）×VRT×109÷(W ×VS÷VE) ÷T=53.59×ΔA÷W

• 通過細菌或細胞量計算:

單位定義: One unit of enzyme activity is defined as that 10 thousand bacteria or cells per minute consumes 1 nmol of NADH.

PFP activity (U/104細胞) =ΔA÷（ε×d）×VRT×109÷(VS×N÷VE) ÷T=53.59×ΔA÷N（104）

• Calculated by serum and other liquids:

單位定義: One unit of enzyme activity is defined as that 1 mL of liquids per minute consumes 1 nmol of NADH.

PFP activity (單位/毫升) =ΔA÷（ε×d）×VRT×109÷VS÷T=53.59×ΔA

虛擬實境測試: total volume of reaction system, 0.001 L;

e: NADH摩爾滅絕係數, 6.22 × 103 L/mol/cm; d: cuvette light path, 1 公分;

VS: added sample volume, 0.1 毫升;

VE: volume of extract solution added, 1 毫升; 時間: 反應時間, 30 分分鐘;

心肺復甦: 樣品蛋白濃度, 毫克/毫升; 瓦: 樣品質量, 0.1 G;

109:轉換因子, 1 mol = 109 nmol N: number of cell

2. Calculated by 96-well UV plate:

Modify d = 1 cm in the above formula to d-0.6 公分 (the light path of a 96-well plate) for calculation.

筆記:

1. The number of samples should not be too large to avoid delaying the enzymatic reaction time.

實驗實例:

1. 拿 0.1 g of bean sprouts and add 1 mL of Extract solution for sample processing. After centrifugation to take the supernatant, 按確定程序進行. CalculateΔA = (A1T-A2T)-(A1B-A2B)= (1.041-0.963)-0=0.078. The enzyme activity is calculated according to the sample mass.

PFP activity (U/g鮮重) =53.59×ΔA÷W=41.8 U/g fresh weight.

相關產品:

BC0990/BC0995植物葉綠素含量測定套件

BC2210/BC2215 Glyceraldehyde-3-phosphate Dehydrogenase(gapdh) 活性測定試劑盒

BC4330/BC4335植物類胡蘿蔔素含量分析套件