BCA蛋白(bái)濃度測定試劑盒（目錄号：RTP7102）

BCA Protein Assay Kit

● 試劑盒内容及保存：

● 儲存條件(jiàn)和效期：

試劑A和試劑B室溫貯存；牛血清白(bái)蛋白(bái)标準溶液-20℃貯存；PBS溶液4℃貯存。本試劑盒有效期1年(nián)。

● 産品簡介：

BCA蛋白(bái)濃度測定試劑盒的原理是蛋白(bái)質分子中肽鍵結構在堿性環境下(xià)能(néng)與Cu²⁺生(shēng)産絡合物(wù)，并将Cu²⁺還(hái)原成Cu⁺，而BCA試劑可以特異性地與Cu⁺結合，形成穩定的有顔色的複合物(wù)，并在562nm處有最大的光(guāng)吸收值，該複合物(wù)顔色的深淺與蛋白(bái)質濃度成正比，可以根據吸收值的大小(xiǎo)來測定蛋白(bái)的含量。

本試劑盒可以檢測500個(gè)樣品（使用微孔闆）或50個(gè)樣品（使用試管）。

● 産品特點：

1. 靈敏度高(gāo)，檢測濃度下(xià)限達到(dào)25μg/ml（在20-1000μg/ml濃度範圍内有較好的線性關系），最小(xiǎo)檢測蛋白(bái)量達到(dào)0.2μg，待測樣品體積為(wèi)1-20μl。

2. BCA法測定蛋白(bái)濃度的最大優點是蛋白(bái)濃度的測定可以耐受高(gāo)濃度的去垢劑，可以兼容樣品中高(gāo)達5%的SDS，5%的Triton X-100，5%的Tween 20, 60, 80。但受螯合劑和略高(gāo)濃度的還(hái)原劑的影響，需确保EDTA低(dī)于10mM，無EGTA，二硫蘇糖醇低(dī)于1mM，β-巯基乙醇低(dī)于0.01%。

● 操作方法

BCA工(gōng)作液配制：

将試劑A和試劑B按照(zhào)體積比50:1比例混合，配成BCA工(gōng)作液。

如，取50ml 試劑A與1ml試劑B混合，配成51 ml BCA工(gōng)作液。兩者混合時會(huì)有沉澱形成，徹底混勻後沉澱消失，溶液應為(wèi)澄清淡藍色溶液。

注：BCA工(gōng)作液室溫可放(fàng)置一(yī)周不失效。

微孔闆測定程序：（工(gōng)作範圍20-2000 μg/ml）

1. 蛋白(bái)标準品配制：室溫完全溶解蛋白(bái)标準品，取20μl 5mg/ml BSA蛋白(bái)标準溶液用PBS溶液稀釋至100μl，使其終濃度為(wèi)1.0 mg/ml。

2. 按照(zhào)下(xià)表配制BSA标準測定溶液： 

3. 将适當體積的待測樣品加入到(dào)微孔闆中，并用PBS補足到(dào)20 μl

4. 向微孔闆中加入200 μl BCA工(gōng)作液，混勻，37℃放(fàng)置30分鍾；

注：也可以室溫放(fàng)置2小(xiǎo)時，或60℃放(fàng)置30分鍾。BCA法測定蛋白(bái)濃度時，顔色會(huì)随著(zhe)時間的延長(cháng)不斷加深。并且顯色反應會(huì)因溫度升高(gāo)而加快。如果濃度較低(dī)，适合在較高(gāo)溫度孵育，或适當延長(cháng)孵育時間。

5. 測定562 nm 處的吸光(guāng)值，并記錄讀(dú)數；以不含BSA 的樣品的光(guāng)吸收值作為(wèi)空白(bái)對照(zhào)。

6. 以A₅₆₂為(wèi)縱坐标，BSA含量為(wèi)橫坐标，繪制标準曲線，計算(suàn)樣品中的蛋白(bái)濃度。如果所得到(dào)的蛋白(bái)濃度不在标準曲線範圍内，請稀釋樣品後重新測定。

試管測定程序：（工(gōng)作範圍20-1000 μg/ml）

1. 蛋白(bái)标準品配制：室溫完全溶解蛋白(bái)标準品，取150μl 5mg/ml BSA蛋白(bái)标準溶液，加入600μl PBS溶液稀釋至750μl，使其終濃度為(wèi)1.0 mg/ml。

2. 按照(zhào)下(xià)表配制BSA标準測定溶液：

3. 将适當體積的待測樣品加入到(dào)試管中，并用PBS補足到(dào)100 μl；

4. 向試管中加入2ml BCA工(gōng)作液，混勻，37℃放(fàng)置30分鍾；

注：也可以室溫放(fàng)置2小(xiǎo)時，或60℃放(fàng)置30分鍾。BCA法測定蛋白(bái)濃度時，顔色會(huì)随著(zhe)時間的延長(cháng)不斷加深。并且顯色反應會(huì)因溫度升高(gāo)而加快。如果濃度較低(dī)，适合在較高(gāo)溫度孵育，或适當延長(cháng)孵育時間。

5. 測定562 nm 處的吸光(guāng)值，并記錄讀(dú)數；以不含BSA 的樣品的光(guāng)吸收值作為(wèi)空白(bái)對照(zhào)。

6. 以A₅₆₂為(wèi)縱坐标，BSA含量為(wèi)橫坐标，繪制标準曲線，計算(suàn)樣品中的蛋白(bái)濃度。如果所得到(dào)的蛋白(bái)濃度不在标準曲線範圍内，請稀釋樣品後重新測定。

● 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(bái)定量試劑盒

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