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BCA蛋白濃度測定試劑盒（目錄號：RTP7102）

BCA Protein Assay Kit

● 試劑盒內容及保存：

● 儲存條件和效期：

試(shi)(shi)劑A和試(shi)(shi)劑B室(shi)溫貯(zhu)(zhu)存；牛血(xue)清白蛋白標(biao)準溶(rong)液-20℃貯(zhu)(zhu)存；PBS溶(rong)液4℃貯(zhu)(zhu)存。本試(shi)(shi)劑盒有效期1年。

● 產品簡介：

BCA蛋白濃度測定試劑盒的原理是蛋白質分子中肽鍵結構在堿性環境下能與Cu²⁺生產絡合物，并將Cu²⁺還原成Cu⁺，而BCA試劑可以特異性地與Cu⁺結(jie)合，形成穩定(ding)的(de)(de)有顏色的(de)(de)復(fu)合物，并在562nm處有最大的(de)(de)光吸收(shou)(shou)值(zhi)，該復(fu)合物顏色的(de)(de)深(shen)淺與蛋(dan)(dan)白(bai)質濃度(du)成正比，可(ke)以根據吸收(shou)(shou)值(zhi)的(de)(de)大小來測定(ding)蛋(dan)(dan)白(bai)的(de)(de)含量。

本試劑盒可以檢測500個樣品（使用微孔板）或50個樣品（使用試管）。

● 產品特點：

1. 靈敏度高，檢測濃(nong)度下限達到25μg/ml（在20-1000μg/ml濃(nong)度范圍(wei)內有(you)較好(hao)的線性關系），最小(xiao)檢測蛋白量達到0.2μg，待測樣品體積為1-20μl。

2. BCA法(fa)測(ce)(ce)定蛋(dan)白濃(nong)度的最大(da)優(you)點是蛋(dan)白濃(nong)度的測(ce)(ce)定可(ke)以耐(nai)受高(gao)濃(nong)度的去垢劑，可(ke)以兼容樣品中(zhong)高(gao)達(da)5%的SDS，5%的Triton X-100，5%的Tween 20, 60, 80。但(dan)受螯合劑和略高(gao)濃(nong)度的還原(yuan)劑的影響，需(xu)確保EDTA低于(yu)10mM，無EGTA，二硫蘇糖醇低于(yu)1mM，β-巰基乙醇低于(yu)0.01%。

● 操作方法

BCA工作液配制：

將試劑A和試劑B按照體積比50:1比例混合，配成BCA工作液。

如，取50ml 試劑A與1ml試劑B混合(he)，配成51 ml BCA工作液(ye)(ye)。兩者混合(he)時會有(you)沉淀(dian)形成，徹底混勻后沉淀(dian)消失，溶液(ye)(ye)應為澄清淡藍(lan)色溶液(ye)(ye)。

注：BCA工作液室溫可放置一周不失效。

微孔板測定程序：（工作范圍20-2000 μg/ml）

1. 蛋(dan)白標(biao)準品配制：室溫完全溶(rong)解蛋(dan)白標(biao)準品，取20μl 5mg/ml BSA蛋(dan)白標(biao)準溶(rong)液用(yong)PBS溶(rong)液稀釋(shi)至(zhi)100μl，使其(qi)終濃度為1.0 mg/ml。

2. 按照下表配制BSA標準測定溶液： 

3. 將適當體(ti)積的待測樣品加入到微孔板中，并用PBS補足到20 μl

4. 向微孔板中加入200 μl BCA工作液，混勻，37℃放置30分鐘；

注：也可以室溫(wen)放置(zhi)2小時，或60℃放置(zhi)30分鐘。BCA法(fa)測(ce)定蛋(dan)白(bai)濃度(du)(du)時，顏(yan)色(se)會隨著時間的延長(chang)不斷(duan)加深。并且顯色(se)反應(ying)會因溫(wen)度(du)(du)升高而加快。如果濃度(du)(du)較低，適合在較高溫(wen)度(du)(du)孵育(yu)，或適當(dang)延長(chang)孵育(yu)時間。

5. 測定562 nm 處的吸(xi)光值，并(bing)記(ji)錄讀數(shu)；以不含(han)BSA 的樣品(pin)的光吸(xi)收值作為空白對照。

6. 以A₅₆₂為縱坐(zuo)標，BSA含量為橫(heng)坐(zuo)標，繪制標準(zhun)曲(qu)(qu)線，計(ji)算樣(yang)品中的蛋白濃度。如果所得(de)到的蛋白濃度不(bu)在(zai)標準(zhun)曲(qu)(qu)線范圍內，請稀(xi)釋(shi)樣(yang)品后(hou)重新測定。

試管測定程序：（工作范圍20-1000 μg/ml）

1. 蛋白標準品配制(zhi)：室溫完全溶(rong)(rong)解蛋白標準品，取150μl 5mg/ml BSA蛋白標準溶(rong)(rong)液，加(jia)入600μl PBS溶(rong)(rong)液稀釋至750μl，使其終濃度為1.0 mg/ml。

 2. 按照下(xia)表(biao)配制(zhi)BSA標準測定(ding)溶液(ye)：

3. 將適當體積的待測樣品加入到試管中，并用PBS補足到100 μl；

4. 向試管中加入2ml BCA工作液，混勻，37℃放置30分鐘；

注：也可以室溫(wen)放置2小(xiao)時(shi)，或60℃放置30分鐘。BCA法測定蛋白(bai)濃度(du)(du)時(shi)，顏色會隨著時(shi)間的延(yan)(yan)長不(bu)斷加(jia)深。并且顯(xian)色反應會因溫(wen)度(du)(du)升高(gao)而加(jia)快。如(ru)果濃度(du)(du)較(jiao)低，適(shi)合在較(jiao)高(gao)溫(wen)度(du)(du)孵育，或適(shi)當(dang)延(yan)(yan)長孵育時(shi)間。

5. 測定562 nm 處的(de)(de)吸光值，并記錄讀數；以(yi)不含BSA 的(de)(de)樣品的(de)(de)光吸收(shou)值作為空白對(dui)照。

6. 以A₅₆₂為縱坐標，BSA含量為橫坐標，繪制(zhi)標準(zhun)曲(qu)(qu)線(xian)，計算樣品(pin)(pin)中(zhong)的(de)蛋白濃度。如果(guo)所(suo)得到的(de)蛋白濃度不在(zai)標準(zhun)曲(qu)(qu)線(xian)范圍內，請稀釋樣品(pin)(pin)后重新測(ce)定。

● References：

1.       Smith, P.K., et al. (1985). Measurement of protein using bicinchoninic acid. Anal. Biochem. 150:76-85.

2.       Wiechelman, K., et al. (1988). Investigation of the bicinchoninic acid protein assay: Identification of the groups responsible for color formation. Anal Biochem. 175:231-7.

3.       Kessler, R. and Fanestil, D. (1986). Interference by lipids in the determination of protein using bicinchoninic acid. Anal. Biochem. 159:138-42.

4.       Brown, R., et al. (1989). Protein measurement using bicinchoninic acid: elimination of interfering substances. Anal. Biochem. 180:136-9. 

BCA蛋白定量試劑盒

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