Western印迹中蛋白质定量分析的替代方法

Western印迹广泛采用显像密度计法对特异性蛋白质表达进行定量,但是仍然存在敏感性的问题尚未解决。同时,昂贵的试剂与复杂的设备限制了其应用。建立了一种改良定量检测法,使用碱性磷酸酶标记的第二抗体,以萘酚磷酸盐和快红作反应底物。吸附膜上的终末显色用有机溶剂洗脱,通过测定光吸收值定量。结果显示该法更简便、快速、经济而不失其敏感性。

Alternative Method for Quantifying Protein Expression in Western Blotting Analysis

Image densitometry has been widely used to quantify specific cellular protein expression in Western blotting analysis, but still there exists an unresolved problem of sensitivity. Furthermore, the use of expen-sive reagents and complex equipment limits its application. Here, we establish an improved method for quantitative examination by using alkaline phosphatase-labeled secondary antibody in combination with substrate compound naphthol AS-MX phosphate and chromagen 4-chloro-2-methylbenzenediazonium salt (fast red TR). The final colorization on blotted membrane is eluted using an organic solvent dimethylsulfoxide (DMSO) and quantified by reading absorptance at 492 nm. Our results prove that this technique is more simplified, fast and economic, without loss of its sensitivity.