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关于Bradford Assay, 你可能不知道的事

发布: 2016-02-16 1,247阅读 221评论

Bradford方法是最常用的蛋白质定量方法之一。其原理很简单，棕色染料考马斯亮蓝G250与蛋白质结合形成蓝色复合物，复合物在595 nm处有最大吸光值。吸光值与蛋白质浓度成正比。配置已知浓度梯度的标准蛋白质溶液，制作标准曲线。根据标准曲线能够测定未知蛋白的浓度。实验操作步骤能够在任何一个公司销售的Bradford assay试剂盒的protocol中找到。在平台回复Bradford可获得来自Sigma，Thermo fisher 以及Biorad公司的Bradford蛋白质定量试剂盒的protocol。但是关于Bradford assay的一些注意事项，我们可能并不清楚。

1利用Bradford方法制作的标准曲线并非是直线

上图标准曲线采用赛默飞Bradford蛋白质检测试剂盒（Cat：23200），BSA作为标准品制作。从图中可知BSA作为标准品制作的标准曲线，线形部分只在0.1-1.4 mg/mL浓度区间内。因此需要将待测蛋白质浓度稀释或浓缩至这一浓度区间，方可利用线形拟合的公式计算蛋白质浓度。另外，有必要在每次实验时都进行标准曲线的制作。

2高浓度去垢剂影响Bradford方法的准确性

对Bradford方法有干扰的试剂及其浓度可以从BioEngX分享的protocol中了解，平台回复Bradford可获得protocol下载链接。这里列举几个常用的试剂及Bradford方法最大兼容浓度：SDS，最大兼容浓度为0.125%; Tween-20(80), 最大兼容浓度0.061%；咪唑（pH 7.0） , 最大兼容浓度200 mM; HEPES（pH 7.5）,最大兼容浓度100 mM。值得一提的是咪唑，利用镍柱纯化蛋白质时，洗脱缓冲溶液中有高浓度的咪唑，因此在利用Bradford对纯化蛋白质进行定量前，必须要对洗脱蛋白质进行脱盐处理。 值得第二提的是，不像BCA方法，Bradford方法与还原性试剂是兼容的。当你需要在蛋白质中添加DTT 等还原性保护试剂时，不要犹豫，Bradford是你绝佳的选择。

3 可利用除了595 nm以外的波长进行检测

Bradford方法能够利用96孔板检测体积低至5 μL的蛋白质浓度，然而很多plate reader不具备595 nm的滤光片。这里要说明的是，蛋白质与染料产生的蓝色复合物可以利用570-610 nm之间的任何波长检测到。只不过，利用除了595 nm以外的波长制作的标准曲线的斜率会比595nm下的标曲斜率小，同时最低检测限也会有所提高。

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