Solarbio乳酸去氫酶 (乳酸脫氫酶) 活性測定試劑盒

乳酸脫氫酶 (乳酸脫氫酶) 活性測定試劑盒

筆記: 測試前取兩個或三個不同的樣本進行預測.

操作設備: 分光光度計/酶標儀

目錄號: BC0685

尺寸:100T/48S

成分:

萃取液: 60 毫升×1. 4℃保存；

試劑一: 7 毫升×1. 4℃保存.

試劑二: 粉末×1. -20℃保存. 工作溶液: dissolve with 1.3 使用前的毫升蒸餾水. It can be divided into tubule after matching, which can be preserved for two weeks at -20℃. Avoid repeated freeze and thaw cycles.

試劑三: 7 毫升×1. 4℃保存.

試劑四: 25 毫升×1. 4℃保存.

Sodium pyruvate standard solution: 1 毫升 (2 微摩爾/毫升) ×1. 4℃保存.

產品描述:

Lactate dehydrogenase (LDH or LD) is the terminal enzyme of the glycolysis pathway which is found in nearly all living cells (動物, 植物, and prokaryotes). LDH catalyzes the conversion of lactate to pyruvic acid and back, as it converts NAD+ to NADH and back.

NAD+ and lactic acid are oxidized to pyruvic acid by the catalysis of LDH. Pyruvate further reacted with 2,4-dinitrophenylhydrazide to form pyruvate dinitrobenzone, which show brown red color in alkaline solution and the color depth is proportional to the concentration of pyruvate.

需要但未提供的試劑和設備:

分光光度計/酶標儀, thermostat water bath, 桌上型離心機, 可調式移液器, 微型玻璃比色皿/96孔平底板, 研缽/均質器, 冰, 蒸餾水.

程式:

我. 樣本 準備:

1. 細菌, 細胞, or tissue sample:

細菌或細胞:

將細菌或細胞收集到離心管中. The liquid in the upper layer is discarded after centrifugation. The ratio of bacteria/cell amount (104): 萃取液體積 (毫升) 是500~1000:1(建議採取約 5 萬個細菌/細胞並添加 1 萃取液毫升數). Bacteria and cell is split by ultrasonic (置於冰上, 200瓦, 工作時間3s, 間隔10秒, 重複 30 次). 離心機在 8000 rpm 4℃ for 10 分分鐘, take the supernatant and put it on ice for test.

組織:

依組織質量的比例進行冰浴均質 (G): 萃取液體積 (毫升) = 1: 5〜10 (it is recommended to take about 0.1 g 組織並加入 1 萃取液毫升數). Ice bath homogenization. 離心機在 8000 rpm 和 4℃ 10 分分鐘, take the supernatant and put it on ice for test.

2. 血清 (電漿)樣本:

Detect sample directly.

二. 程式:

1. Preheat the Spectrophotometer/Microplate reader 30 分分鐘, 調整波長至 450 奈米, 用蒸餾水調零.
2. Sodium pyruvate standard solution: 拿 100 µL 標準溶液, dilute to 1, 5, 0.25, 0.125, 0 微摩爾/毫升, and use 2, 1, 0.5, 0.25, 0.125, 0 μmol/mL as standard curve.

3. Sample Test

Mixed thoroughly, place at room temperature for 3 分分鐘. 拿 200 μL of reaction solution in micro glass cuvette/96 well flat-bottom plate, measured the absorbance at 450 奈米, ΔA=AT-AC. Each test tube should set a control tube.

三、. 乳酸脫氫酶 計算.

1. Sample Sodium pyruvate content
2. Set the standard curve, y-axis as the standard concentration, 微摩爾/毫升, x-axis as the 450 nm absorption. Put ΔA(X) into standard curve, calculate y(微摩爾/毫升)
3. 血清 (電漿) sample LDH activity

單位定義: One unit of enzyme activity is defined as the amount of enzyme catalyzes the producing of 1 nmol of pyruvic acid per minute, every milliliter of serum.

乳酸脫氫酶(單位/毫升)=y÷T×103=66.7×y

4. 組織, bacteria or cultured cells LDH activity

A. 蛋白質濃度:

單位定義: One unit of enzyme activity is defined as the amount of enzyme catalyzes the producing of 1 nmol of pyruvic acid per minute, 每毫克蛋白質.

乳酸脫氫酶(U/毫克蛋白質)= y÷T÷Cpr×103 =66.7×y÷Cpr

乙. 樣品重量

單位定義: One unit of enzyme activity is defined as the amount of enzyme catalyzes the producing of 1 nmol of pyruvic acid per minute, in every gram of tissue. 乳酸脫氫酶(U/g)= y÷T÷W×103 =666.7×y

C. 細胞數量

單位定義: One unit of enzyme activity is defined as the amount of enzyme catalyzes the producing of 1 nmol of pyruvic acid per minute every 10000 細胞.

乳酸脫氫酶(U/104 cell)= y÷T÷500×103 =0.133×y

VS: Supernate volume (毫升), 0.01 毫升; 電壓: 萃取液體積, 1 毫升; 時間: 反應時間, 15 分分鐘;

心肺復甦: 樣品蛋白濃度, 毫克/毫升; 瓦: 樣品重量, 0.1 G;

500: 細菌或細胞總數, 5 百萬; 103: 1 μmol/mL=103 nmol/mL.

參考

[1] Huang P H, Fu L C, Huang C S, 等人. The uptake of oligogalacturonide and its effect on growth inhibition, lactate dehydrogenase activity and galactin-3 release of human cancer cells[J]. Food chemistry, 2012, 132(4): 1987-1995.

相關產品

BC0740/BC0745 Hexokinase(HK) 活性測定試劑盒

BC0540/BC0545 Pyruvate Kinase(PK) 活性測定試劑盒

BC2250/BC2255 Phosphoglycerate Kinase(PGK) 活性測定試劑盒

BC2270/BC2275 Fructose-bisphosphate aldolase(FBA) 活性測定試劑盒