Phos-tag SDS-PAGE followed by LC-MS/MS analysis of in vitro phosphorylated Nab2, Foxk1, and HURP

Purified GST-fused Nab2, Foxk1, and HURP were incubated with ATP in the presence or absence of His-tagged active ERK in vitro, separated by Phos-tag SDS-PAGE, and visualized by CBB staining. The most shifted regions of protein bands on the Phos-tag-containing gels were excised, digested with trypsin, and analyzed by LC-MS/MS.

将纯化的谷胱甘肽S-转移酶（GST）融合蛋白Nab2、Foxk1及HURP在体外于含有或不含组氨酸标签（His-tag）的活性ERK的体系中与ATP共同孵育，随后通过Phos-tag SDS-聚丙烯酰胺凝胶电泳进行分离，并经考马斯亮蓝（CBB）染色显影。将含Phos-tag的凝胶上蛋白条带迁移率偏移最为显著的区域切下，用胰蛋白酶进行酶解，随后通过液相色谱-串联质谱（LC-MS/MS）进行分析。