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        線粒體檸檬酸含量測試定試劑盒 微量法

測定意義：CA是線粒體三羧酸(suan)循環的(de)*個中(zhong)間產物，由(you)檸檬酸(suan)合酶催化乙酰CoA與草酰乙酸(suan)合成，其(qi)含(han)量是三羧酸(suan)循環強度的(de)主要指(zhi)標之(zhi)一。

測定原理：CA在檸檬酸(suan)(suan)(suan)裂解酶(mei)的作用下，生(sheng)成(cheng)α-酮(tong)酸(suan)(suan)(suan)（草(cao)酰(xian)乙酸(suan)(suan)(suan)）；在弱酸(suan)(suan)(suan)性條件下，α-酮(tong)酸(suan)(suan)(suan)進一步與苯(ben)肼反應，生(sheng)成(cheng)相應的α-酮(tong)酸(suan)(suan)(suan)苯(ben)腙(zong)；α-酮(tong)酸(suan)(suan)(suan)苯(ben)腙(zong)在330nm處(chu)有吸收峰，該波長下吸光(guang)度的變化程(cheng)度可反映出CA的含量(liang)。

需要的儀器(qi)，耗(hao)材:可見分光(guang)光(guang)度計/酶(mei)標(biao)儀、水浴鍋、可調(diao)式移(yi)液槍、微量石英比色皿/96孔(kong)板、研缽、蒸餾水。 

      ;    線粒體檸檬酸含量測試定試劑盒 微量法

試劑組成(cheng)和配制：酸性(xing)提取(qu)(qu)液(ye)(ye)：液(ye)(ye)體 100mL×1 瓶(ping)(ping)(ping)，4℃保存(cun)(cun)(cun)。堿(jian)性(xing)提取(qu)(qu)液(ye)(ye)：液(ye)(ye)體 100mL×1 瓶(ping)(ping)(ping)，4℃保存(cun)(cun)(cun)。試劑一：液(ye)(ye)體 6mL×1 瓶(ping)(ping)(ping)，4℃保存(cun)(cun)(cun)。試劑二：液(ye)(ye)體 2mL×1 瓶(ping)(ping)(ping)，4℃保存(cun)(cun)(cun)。試劑三：粉劑×1 瓶(ping)(ping)(ping)，4℃保存(cun)(cun)(cun)；臨用(yong)前加入(ru) 6mL 蒸(zheng)餾水充分溶解待用(yong)；用(yong)不(bu)完的(de)試劑 4℃保存(cun)(cun)(cun)；標準(zhun)液(ye)(ye)：液(ye)(ye)體 1mL×1 支，10μmol/mL 檸檬酸標準(zhun)液(ye)(ye)，4℃保存(cun)(cun)(cun)。

線(xian)粒體中(zhong)檸檬(meng)酸提(ti)(ti)取(qu)(qu)(qu)：稱 0.05~0.1g 樣(yang)(yang)品（建(jian)議稱 0.1g 樣(yang)(yang)本(ben)(ben)），加(jia)入 0.5mL 酸性提(ti)(ti)取(qu)(qu)(qu)液(ye)，冰上(shang)充(chong)分研磨(mo)，600g/min 4℃ 離心 5min；取(qu)(qu)(qu)上(shang)清(qing)(qing)另一(yi)(yi) EP 管(guan)(guan)(guan)(guan)(guan)(guan)中(zhong)，11000g/min 4℃離心 10min，棄上(shang)清(qing)(qing)（取(qu)(qu)(qu) 300μL 該(gai)上(shang)清(qing)(qing)液(ye)和(he) 300μL 堿性提(ti)(ti)取(qu)(qu)(qu)液(ye)中(zhong)和(he)后可用(yong)于細胞(bao)質 CA 含(han)量(liang)(liang)測(ce)(ce)(ce)(ce)(ce)定(ding)(ding)(ding)(ding)(ding)）；沉淀即線(xian)粒體，向沉淀中(zhong)加(jia)入 0.5mL 酸性提(ti)(ti)取(qu)(qu)(qu)液(ye)，充(chong)分懸浮溶解，超聲波破碎(sui)（功(gong)率 20％，超聲 3 秒(miao)，間隔 10 秒(miao)，重復 30 次），取(qu)(qu)(qu)此溶液(ye) 300μL 和(he) 300μL 堿性提(ti)(ti)取(qu)(qu)(qu)液(ye)中(zhong)和(he)，混(hun)勻(yun)，置冰上(shang)待測(ce)(ce)(ce)(ce)(ce)（不可用(yong)于蛋(dan)(dan)白(bai)(bai)質含(han)量(liang)(liang)測(ce)(ce)(ce)(ce)(ce)定(ding)(ding)(ding)(ding)(ding)）。測(ce)(ce)(ce)(ce)(ce)定(ding)(ding)(ding)(ding)(ding)步驟： 1、分光光度計(ji)(ji)或酶標(biao)儀預熱(re) 30 min 以(yi)上(shang)，調節波長到 330nm，蒸(zheng)餾(liu)水(shui)調零。 2、試(shi)(shi)劑(ji)一(yi)(yi)、二和(he)三 37℃預熱(re) 10min。 3、樣(yang)(yang)本(ben)(ben)測(ce)(ce)(ce)(ce)(ce)定(ding)(ding)(ding)(ding)(ding)：空(kong)(kong)(kong)白(bai)(bai)管(guan)(guan)(guan)(guan)(guan)(guan)和(he)標(biao)準(zhun)(zhun)管(guan)(guan)(guan)(guan)(guan)(guan)只需要各(ge)做一(yi)(yi)個。試(shi)(shi)劑(ji)名(ming)稱(μL) 空(kong)(kong)(kong)白(bai)(bai)管(guan)(guan)(guan)(guan)(guan)(guan) 標(biao)準(zhun)(zhun)管(guan)(guan)(guan)(guan)(guan)(guan) 測(ce)(ce)(ce)(ce)(ce)定(ding)(ding)(ding)(ding)(ding)管(guan)(guan)(guan)(guan)(guan)(guan)試(shi)(shi)劑(ji)一(yi)(yi) 60 60 60 蒸(zheng)餾(liu)水(shui) 60 標(biao)準(zhun)(zhun)液(ye) 60 樣(yang)(yang)本(ben)(ben) 60 試(shi)(shi)劑(ji)二 20 20 20 試(shi)(shi)劑(ji)三 60 60 60 充(chong)分混(hun)勻(yun)，330nm 立即測(ce)(ce)(ce)(ce)(ce)定(ding)(ding)(ding)(ding)(ding)初始吸光值 A1 和(he) 37℃孵(fu)育 30min 后的吸光值 A2，ΔA=A2 –A1。檸檬(meng)酸含(han)量(liang)(liang)計(ji)(ji)算：（1）按(an)蛋(dan)(dan)白(bai)(bai)濃(nong)度計(ji)(ji)算檸檬(meng)酸含(han)量(liang)(liang)(μmol/mg prot)=[C 標(biao)準(zhun)(zhun)管(guan)(guan)(guan)(guan)(guan)(guan)×(ΔA 測(ce)(ce)(ce)(ce)(ce)定(ding)(ding)(ding)(ding)(ding)管(guan)(guan)(guan)(guan)(guan)(guan)－ΔA 空(kong)(kong)(kong)白(bai)(bai)管(guan)(guan)(guan)(guan)(guan)(guan))÷(ΔA 標(biao)準(zhun)(zhun)管(guan)(guan)(guan)(guan)(guan)(guan)－ΔA 空(kong)(kong)(kong)白(bai)(bai)管(guan)(guan)(guan)(guan)(guan)(guan)) ×V 樣(yang)(yang)]÷（V 樣(yang)(yang)÷Cpr）=10×(ΔA 測(ce)(ce)(ce)(ce)(ce)定(ding)(ding)(ding)(ding)(ding)管(guan)(guan)(guan)(guan)(guan)(guan)－ΔA 空(kong)(kong)(kong)白(bai)(bai)管(guan)(guan)(guan)(guan)(guan)(guan))÷(ΔA 標(biao)準(zhun)(zhun)管(guan)(guan)(guan)(guan)(guan)(guan)－ΔA 空(kong)(kong)(kong)白(bai)(bai)管(guan)(guan)(guan)(guan)(guan)(guan))÷Cpr 蛋(dan)(dan)白(bai)(bai)質含(han)量(liang)(liang)需要另外測(ce)(ce)(ce)(ce)(ce)定(ding)(ding)(ding)(ding)(ding)。（2）按(an)樣(yang)(yang)本(ben)(ben)鮮(xian)重計(ji)(ji)算檸檬(meng)酸含(han)量(liang)(liang)(μ mol/g鮮(xian)重)=[C標(biao)準(zhun)(zhun)管(guan)(guan)(guan)(guan)(guan)(guan)×(ΔA測(ce)(ce)(ce)(ce)(ce)定(ding)(ding)(ding)(ding)(ding)管(guan)(guan)(guan)(guan)(guan)(guan)－ΔA空(kong)(kong)(kong)白(bai)(bai)管(guan)(guan)(guan)(guan)(guan)(guan))÷(ΔA標(biao)準(zhun)(zhun)管(guan)(guan)(guan)(guan)(guan)(guan)－ΔA空(kong)(kong)(kong)白(bai)(bai)管(guan)(guan)(guan)(guan)(guan)(guan)) ×V 樣(yang)(yang)]÷（W×V 樣(yang)(yang)÷V 樣(yang)(yang)總）=10×(ΔA 測(ce)(ce)(ce)(ce)(ce)定(ding)(ding)(ding)(ding)(ding)管(guan)(guan)(guan)(guan)(guan)(guan)－ΔA 空(kong)(kong)(kong)白(bai)(bai)管(guan)(guan)(guan)(guan)(guan)(guan))÷(ΔA 標(biao)準(zhun)(zhun)管(guan)(guan)(guan)(guan)(guan)(guan)－ΔA 空(kong)(kong)(kong)白(bai)(bai)管(guan)(guan)(guan)(guan)(guan)(guan))÷W C 標(biao)準(zhun)(zhun)管(guan)(guan)(guan)(guan)(guan)(guan)：標(biao)準(zhun)(zhun)液(ye)濃(nong)度，10μmol/mL； V 樣(yang)(yang)：加(jia)入反應體系中(zhong)樣(yang)(yang)本(ben)(ben)體積(ji)：0.06mL；V 樣(yang)(yang)總：加(jia)入提(ti)(ti)取(qu)(qu)(qu)液(ye)體積(ji)：1mL；Cpr：樣(yang)(yang)品蛋(dan)(dan)白(bai)(bai)濃(nong)度，mg/mL；W：樣(yang)(yang)本(ben)(ben)質量(liang)(liang)，g。注意(yi)：檢測(ce)(ce)(ce)(ce)(ce)限為 10nmol/mg prot 或 1μmol/g 鮮(xian)重。

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