BCA蛋白濃度測定試劑盒（目錄號：RTP7102）

BCA Protein Assay Kit

● 試劑盒內(nèi)容及保存：

● 儲存條件和效期：

試劑A和試劑B室溫貯存；牛血清白蛋白標準溶液-20℃貯存；PBS溶液4℃貯存。本試劑盒有效期1年。

● 產(chǎn)品簡介：

BCA蛋白濃度測定試劑盒的原理是蛋白質(zhì)分子中肽鍵結(jié)構(gòu)在堿性環(huán)境下能與Cu²⁺生產(chǎn)絡(luò)合物，并將Cu²⁺還原成Cu⁺，而BCA試劑可以特異性地與Cu⁺結(jié)合，形成穩(wěn)定的有顏色的復(fù)合物，并在562nm處有最大的光吸收值，該復(fù)合物顏色的深淺與蛋白質(zhì)濃度成正比，可以根據(jù)吸收值的大小來測定蛋白的含量。

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(chǎn)品特點：

1. 靈敏度高，檢測濃度下限達到25μg/ml（在20-1000μg/ml濃度范圍內(nèi)有較好的線性關(guān)系），最小檢測蛋白量達到0.2μg，待測樣品體積為1-20μl。

2. BCA法測定蛋白濃度的最大優(yōu)點是蛋白濃度的測定可以耐受高濃度的去垢劑，可以兼容樣品中高達5%的SDS，5%的Triton X-100，5%的Tween 20, 60, 80。但受螯合劑和略高濃度的還原劑的影響，需確保EDTA低于10mM，無EGTA，二硫蘇糖醇低于1mM，β-巰基乙醇低于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，配成51 ml BCA工作液。兩者混合時會有沉淀形成，徹底混勻后沉淀消失，溶液應(yīng)為澄清淡藍色溶液。

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. 蛋白標準品配制：室溫完全溶解蛋白標準品，取20μl 5mg/ml BSA蛋白標準溶液用PBS溶液稀釋至100μl，使其終濃度為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：也可以室溫放置2小時，或60℃放置30分鐘。BCA法測定蛋白濃度時，顏色會隨著時間的延長不斷加深。并且顯色反應(yīng)會因溫度升高而加快。如果濃度較低，適合在較高溫度孵育，或適當延長孵育時間。

5. 測定562 nm 處的吸光值，并記錄讀數(shù)；以不含BSA 的樣品的光吸收值作為空白對照。

6. 以A₅₆₂為縱坐標，BSA含量為橫坐標，繪制標準曲線，計算樣品中的蛋白濃度。如果所得到的蛋白濃度不在標準曲線范圍內(nèi)，請稀釋樣品后重新測定。

試管測定程序：（工作范圍20-1000 μg/ml）

1. 蛋白標準品配制：室溫完全溶解蛋白標準品，取150μl 5mg/ml BSA蛋白標準溶液，加入600μl PBS溶液稀釋至750μl，使其終濃度為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：也可以室溫放置2小時，或60℃放置30分鐘。BCA法測定蛋白濃度時，顏色會隨著時間的延長不斷加深。并且顯色反應(yīng)會因溫度升高而加快。如果濃度較低，適合在較高溫度孵育，或適當延長孵育時間。

5. 測定562 nm 處的吸光值，并記錄讀數(shù)；以不含BSA 的樣品的光吸收值作為空白對照。

6. 以A₅₆₂為縱坐標，BSA含量為橫坐標，繪制標準曲線，計算樣品中的蛋白濃度。如果所得到的蛋白濃度不在標準曲線范圍內(nèi)，請稀釋樣品后重新測定。

● 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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