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乙酰輔酶A羧化酶試劑盒 微量法

樣本的前處理

1、細菌(jun)(jun)或培養細胞：先收集細菌(jun)(jun)或細胞到離心(xin)管內，離心(xin)后棄上(shang)清；按照細菌(jun)(jun)或細胞數量（104個）：提取(qu)液體積（mL）為 500~1000：1 的比例（建議 500 萬細菌(jun)(jun)或細胞加入 1mL 提取(qu)液），超聲波破碎細菌(jun)(jun)或細胞（冰浴，功率 20％或 200W，超聲 3s，間隔 10s，重復 30 次）； 8000g 4℃離心(xin) 10min，取(qu)上(shang)清，置冰上(shang)待測。

2、組織：按照組織質量（g）：提取(qu)(qu)液體積(mL)為(wei) 1：5~10 的比例(li)（建議稱取(qu)(qu)約 0.1g 組織，加入 1mL 提取(qu)(qu)液），進行冰(bing)浴勻漿。8000g 4℃離心(xin) 10min，取(qu)(qu)上清，置冰(bing)上待測。

測定步驟

1、 分(fen)光光度計或酶標(biao)儀(yi)預熱(re) 30min 以上，調節波長 660nm，蒸餾水調零。

2、 酶(mei)促(cu)反(fan)應試劑的配制和預熱(re)：在(zai)試劑二(er)瓶(ping)(ping)中加入 2.5mL 試劑一(yi)，充分(fen)溶解(jie)(jie)混勻；在(zai)試劑三瓶(ping)(ping)中加入 2mL 蒸餾水，充分(fen)溶解(jie)(jie)混勻；將試劑一(yi)、二(er)、三在(zai) 37℃(哺乳(ru)動物)或 25℃ (其它物種(zhong))預熱(re) 10 分(fen)鐘(zhong)。

3、定(ding)(ding)磷(lin)試(shi)劑(ji)的(de)配制(zhi)：按 H2O: 試(shi)劑(ji)四(si):試(shi)劑(ji)五:試(shi)劑(ji)六=2:1:1:1 的(de)比例配制(zhi)，配好的(de)定(ding)(ding)磷(lin)試(shi)劑(ji) 應(ying)為淺(qian)黃色，若無色則試(shi)劑(ji)失效，若是藍(lan)色則為磷(lin)污染，定(ding)(ding)磷(lin)劑(ji)現用現配。注(zhu)意：配試(shi)劑(ji)用新的(de)燒杯、玻(bo)棒和(he)玻(bo)璃移液器(qi)，也可以用一次性塑料(liao)器(qi)皿，避(bi)免磷(lin)污染。

4、0.5μmol/mL 標(biao)準磷應用液配(pei)制：將試(shi)(shi)劑七 20 倍稀(xi)釋(shi)，即取 0.5mL 試(shi)(shi)劑七加 9.5 蒸餾水，充(chong)分(fen)混(hun)勻。

乙酰輔酶A羧化酶試劑盒 微量法

ACC 活性計(ji)(ji)算(suan)(suan)： 1、按組織(zhi)蛋白(bai)(bai)濃度(du)(du)計(ji)(ji)算(suan)(suan)：定(ding)義：每小時每毫(hao)克組織(zhi)蛋白(bai)(bai)產生(sheng) 1μmol 無機(ji)磷的(de)量為(wei)一(yi)(yi)個 ACC 活力(li)單位(wei)。 ACC (μmol/h/mg prot)=(C 標(biao)(biao)準(zhun)(zhun)(zhun)管(guan)(guan)(guan)(guan)(guan)(guan)(guan)(guan)(guan)×V 總(zong))×(A 測(ce)定(ding)管(guan)(guan)(guan)(guan)(guan)(guan)(guan)(guan)(guan)-A 對(dui)(dui)照(zhao)(zhao)(zhao)(zhao)管(guan)(guan)(guan)(guan)(guan)(guan)(guan)(guan)(guan))÷(A 標(biao)(biao)準(zhun)(zhun)(zhun)管(guan)(guan)(guan)(guan)(guan)(guan)(guan)(guan)(guan)-A 空(kong)白(bai)(bai)管(guan)(guan)(guan)(guan)(guan)(guan)(guan)(guan)(guan))÷ (V 樣(yang)(yang)(yang)×Cpr)÷T=10×(A 測(ce)定(ding)管(guan)(guan)(guan)(guan)(guan)(guan)(guan)(guan)(guan)-A 對(dui)(dui)照(zhao)(zhao)(zhao)(zhao)管(guan)(guan)(guan)(guan)(guan)(guan)(guan)(guan)(guan))÷(A 標(biao)(biao)準(zhun)(zhun)(zhun)管(guan)(guan)(guan)(guan)(guan)(guan)(guan)(guan)(guan)-A 空(kong)白(bai)(bai)管(guan)(guan)(guan)(guan)(guan)(guan)(guan)(guan)(guan))÷Cpr 2、按樣(yang)(yang)(yang)本鮮重計(ji)(ji)算(suan)(suan)：定(ding)義：每小時每 g 組織(zhi)產生(sheng) 1μmol 無機(ji)磷的(de)量為(wei)一(yi)(yi)個 ACC 活力(li)單位(wei)。 ACC (μmol/h /g 鮮重)=(C 標(biao)(biao)準(zhun)(zhun)(zhun)管(guan)(guan)(guan)(guan)(guan)(guan)(guan)(guan)(guan)×V 總(zong))×(A 測(ce)定(ding)管(guan)(guan)(guan)(guan)(guan)(guan)(guan)(guan)(guan)-A 對(dui)(dui)照(zhao)(zhao)(zhao)(zhao)管(guan)(guan)(guan)(guan)(guan)(guan)(guan)(guan)(guan))÷(A 標(biao)(biao)準(zhun)(zhun)(zhun)管(guan)(guan)(guan)(guan)(guan)(guan)(guan)(guan)(guan)-A 空(kong)白(bai)(bai)管(guan)(guan)(guan)(guan)(guan)(guan)(guan)(guan)(guan)）×V 總(zong)÷ (W× V 樣(yang)(yang)(yang)÷V 樣(yang)(yang)(yang)總(zong))÷T=10×(A 測(ce)定(ding)管(guan)(guan)(guan)(guan)(guan)(guan)(guan)(guan)(guan)-A 對(dui)(dui)照(zhao)(zhao)(zhao)(zhao)管(guan)(guan)(guan)(guan)(guan)(guan)(guan)(guan)(guan))÷(A 標(biao)(biao)準(zhun)(zhun)(zhun)管(guan)(guan)(guan)(guan)(guan)(guan)(guan)(guan)(guan)-A 空(kong)白(bai)(bai)管(guan)(guan)(guan)(guan)(guan)(guan)(guan)(guan)(guan))÷W 3、 按細(xi)(xi)(xi)菌(jun)或細(xi)(xi)(xi)胞(bao)密度(du)(du)計(ji)(ji)算(suan)(suan)：定(ding)義：每小時每 500 萬細(xi)(xi)(xi)菌(jun)或細(xi)(xi)(xi)胞(bao)產生(sheng) 1μmol 無機(ji)磷的(de)量為(wei)一(yi)(yi)個 ACC 活力(li)單位(wei)。 ACC (U/104 cell)=(C 標(biao)(biao)準(zhun)(zhun)(zhun)管(guan)(guan)(guan)(guan)(guan)(guan)(guan)(guan)(guan)×V 總(zong))×(A 測(ce)定(ding)管(guan)(guan)(guan)(guan)(guan)(guan)(guan)(guan)(guan)-A 對(dui)(dui)照(zhao)(zhao)(zhao)(zhao)管(guan)(guan)(guan)(guan)(guan)(guan)(guan)(guan)(guan))÷(A 標(biao)(biao)準(zhun)(zhun)(zhun)管(guan)(guan)(guan)(guan)(guan)(guan)(guan)(guan)(guan)-A 空(kong)白(bai)(bai)管(guan)(guan)(guan)(guan)(guan)(guan)(guan)(guan)(guan)）×V 總(zong)÷ (500× V 樣(yang)(yang)(yang)÷V 樣(yang)(yang)(yang)總(zong))÷T=0.02×(A 測(ce)定(ding)管(guan)(guan)(guan)(guan)(guan)(guan)(guan)(guan)(guan)-A 對(dui)(dui)照(zhao)(zhao)(zhao)(zhao)管(guan)(guan)(guan)(guan)(guan)(guan)(guan)(guan)(guan))÷(A 標(biao)(biao)準(zhun)(zhun)(zhun)管(guan)(guan)(guan)(guan)(guan)(guan)(guan)(guan)(guan)-A 空(kong)白(bai)(bai)管(guan)(guan)(guan)(guan)(guan)(guan)(guan)(guan)(guan)) C 標(biao)(biao)準(zhun)(zhun)(zhun)管(guan)(guan)(guan)(guan)(guan)(guan)(guan)(guan)(guan)：標(biao)(biao)準(zhun)(zhun)(zhun)管(guan)(guan)(guan)(guan)(guan)(guan)(guan)(guan)(guan)濃度(du)(du)，0.5μmol/mL；V 總(zong)：酶促反(fan)應(ying)(ying)總(zong)體(ti)積(ji)，0.1mL；V 樣(yang)(yang)(yang)：加入樣(yang)(yang)(yang)本體(ti)積(ji)，0.01mL ；V 樣(yang)(yang)(yang)總(zong)：加入提取液體(ti)積(ji)，1mL；T：反(fan)應(ying)(ying)時間，0.5 小時；Cpr：樣(yang)(yang)(yang)本蛋白(bai)(bai)質濃度(du)(du)，mg/mL；W：樣(yang)(yang)(yang)本鮮重，g；500：細(xi)(xi)(xi)菌(jun)或細(xi)(xi)(xi)胞(bao)總(zong)數(shu)，500 萬。

ACC 能夠(gou)催化乙(yi)酰(xian)輔(fu)酶(mei)(mei) A、NaHCO3和 ATP 生成丙(bing)二酰(xian)輔(fu)酶(mei)(mei) A、ATP 和無機磷，通過鉬酸(suan)銨定(ding)磷法測(ce)定(ding)無機磷的增(zeng)加量來測(ce)定(ding) ACC 活性(xing)。