BCA蛋白浓度测定试剂盒（目录号：RTP7102）

BCA Protein Assay Kit

● 试剂盒内容及保存：

● 储存条件和效期：

试剂A和试剂B室温贮存；牛血清白蛋白标准溶液-20℃贮存；PBS溶液4℃贮存。本试剂盒有效期1年。

● 产品简介：

BCA蛋白浓度测定试剂盒的原理是蛋白质分子中肽键结构在碱性环境下能与Cu²⁺生产络合物，并将Cu²⁺还原成Cu⁺，而BCA试剂可以特异性地与Cu⁺结合，形成稳定的有颜色的复合物，并在562nm处有最大的光吸收值，该复合物颜色的深浅与蛋白质浓度成正比，可以根据吸收值的大小来测定蛋白的含量。

本试剂盒可以检测500个样品（使用微孔板）或50个样品（使用试管）。

● 产品特点：

1. 灵敏度高，检测浓度下限达到25μg/ml（在20-1000μg/ml浓度范围内有较好的线性关系），最小检测蛋白量达到0.2μg，待测样品体积为1-20μl。

2. BCA法测定蛋白浓度的最大优点是蛋白浓度的测定可以耐受高浓度的去垢剂，可以兼容样品中高达5%的SDS，5%的Triton X-100，5%的Tween 20, 60, 80。但受螯合剂和略高浓度的还原剂的影响，需确保EDTA低于10mM，无EGTA，二硫苏糖醇低于1mM，β-巯基乙醇低于0.01%。

● 操作方法

BCA工作液配制：

将试剂A和试剂B按照体积比50:1比例混合，配成BCA工作液。

如，取50ml 试剂A与1ml试剂B混合，配成51 ml BCA工作液。两者混合时会有沉淀形成，彻底混匀后沉淀消失，溶液应为澄清淡蓝色溶液。

注：BCA工作液室温可放置一周不失效。

微孔板测定程序：（工作范围20-2000 μg/ml）

1. 蛋白标准品配制：室温完全溶解蛋白标准品，取20μl 5mg/ml BSA蛋白标准溶液用PBS溶液稀释至100μl，使其终浓度为1.0 mg/ml。

2. 按照下表配制BSA标准测定溶液： 

3. 将适当体积的待测样品加入到微孔板中，并用PBS补足到20 μl

4. 向微孔板中加入200 μl BCA工作液，混匀，37℃放置30分钟；

注：也可以室温放置2小时，或60℃放置30分钟。BCA法测定蛋白浓度时，颜色会随着时间的延长不断加深。并且显色反应会因温度升高而加快。如果浓度较低，适合在较高温度孵育，或适当延长孵育时间。

5. 测定562 nm 处的吸光值，并记录读数；以不含BSA 的样品的光吸收值作为空白对照。

6. 以A₅₆₂为纵坐标，BSA含量为横坐标，绘制标准曲线，计算样品中的蛋白浓度。如果所得到的蛋白浓度不在标准曲线范围内，请稀释样品后重新测定。

试管测定程序：（工作范围20-1000 μg/ml）

1. 蛋白标准品配制：室温完全溶解蛋白标准品，取150μl 5mg/ml BSA蛋白标准溶液，加入600μl PBS溶液稀释至750μl，使其终浓度为1.0 mg/ml。

2. 按照下表配制BSA标准测定溶液：

3. 将适当体积的待测样品加入到试管中，并用PBS补足到100 μl；

4. 向试管中加入2ml BCA工作液，混匀，37℃放置30分钟；

注：也可以室温放置2小时，或60℃放置30分钟。BCA法测定蛋白浓度时，颜色会随着时间的延长不断加深。并且显色反应会因温度升高而加快。如果浓度较低，适合在较高温度孵育，或适当延长孵育时间。

5. 测定562 nm 处的吸光值，并记录读数；以不含BSA 的样品的光吸收值作为空白对照。

6. 以A₅₆₂为纵坐标，BSA含量为横坐标，绘制标准曲线，计算样品中的蛋白浓度。如果所得到的蛋白浓度不在标准曲线范围内，请稀释样品后重新测定。

● 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蛋白定量试剂盒

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