Physical Biochemistry, Department of Chemistry, Technische Universität Darmstadt, Darmstadt, Germany.
Electrophoresis. 2009 Oct;30(20):3622-5. doi: 10.1002/elps.200900114.
Native electrophoresis is a powerful tool for the separation of intact protein complexes. By incubating such gels in a suitable reaction solution, specific enzyme activities can be screened comprehensively. The recent standard procedure for determination of ATP hydrolysis activity in blue or clear native gels is based on formation of a lead phosphate precipitate. The resulting white bands are challenging for detection and documentation of low activities. For the analysis of photosynthetic ATP synthases, the method has to be adapted to deregulate the inhibition of latent ATPase functions. Therefore, we introduced an incubation of gels in detergent solution, whereby taurodeoxycholate turned out to be the most efficient activator. In order to detect low ATPase activities, a short additional incubation step subsequent to the formation of lead phosphate is recommended. By adding ammonium sulfide, the white bands are converted into brownish-black bands of lead sulfide. Our new procedure sustains the linear quantitation range of the original lead phosphate protocol and moreover expands the detection limit.
Native electrophoresis 是一种强大的工具，可用于分离完整的蛋白质复合物。通过将这些凝胶在合适的反应溶液中孵育，可以全面筛选特定的酶活性。最近在蓝色或透明天然凝胶中测定 ATP 水解活性的标准程序基于形成磷酸铅沉淀。由此产生的白色条带对于检测和记录低活性具有挑战性。对于光合 ATP 合酶的分析，该方法必须进行调整以解除潜在 ATP 酶功能的抑制。因此，我们引入了在去污剂溶液中孵育凝胶的方法，其中牛磺脱氧胆酸盐被证明是最有效的激活剂。为了检测低 ATP 酶活性，建议在形成磷酸铅后进行短暂的额外孵育步骤。通过添加硫化铵，白色条带会转化为硫化铅的棕黑色条带。我们的新程序维持了原始磷酸铅方案的线性定量范围，并且还扩展了检测限。
