The phosphorylation dynamics of MEK1 in human cells was characterized by using the phosphate affinity electrophoresis technique, Phos-tag sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Phos-tag SDS-PAGE). In this reference, the researchers found that multiple variants of MEK1 with different phosphorylation states are constitutively present in typical human cells (3).
The authors describe phosphorylation profiling of various MEK1 mutants with mutations identified in CFC syndrome, sporadic cancers or MEK-inhibitor-resistant cancer cells. They used Phos-tag SDS-PAGE to investigate the relationships between kinase activity or drug efficacy and the characteristics of individual mutations in the MEK1- coding gene.
A Flexi® HaloTag® clone pFN21AE0668, which is suitable for expression of N-terminal HaloTag®-fused MEK1 in mammalian cells, was prepared from Escherichia coli. Mutations were introduced into the pFN21AE0668 vector. Phos-tag SDS-PAGE was performed on each of the mutations. MEK1 kinase activity was determined by pulldown of HaloTag®-fused proteins using Magne® HaloTag® Beads (Cat.# G7281, G7282) and incubating in the presence of APT and inactive ERK1 (3).