Development of a Plate-Based Assay Platform to Monitor Cellular SHP2 Phosphatase Activity During Erythroid Differentiation

摘要

In light of the growing interest in understanding the roles of protein tyrosine phosphatases (PTPs) in the complicated signaling pathways, convenient methods to profile and analyze PTP activities in various cellular processes are in high demand. We previously demonstrated a two-step assay platform that combined the use of a universal activity-based probe with a PTP-specific antibody, by which the designated PTP activity in a cellular event could be successfully determined. Here, we describe an improved plate-based assay platform by replacing the immunoprecipitation step with a simpler immunocapture step using an antibody on a plate. An assay protocol targeting SHP2 activity was thus established and a universal probe with a sulforhodamine B tag was used to afford a direct fluorescent readout. The protocol was employed for the monitoring of SHP2 activities during K562 erythroid differentiation. Comparative study using the previous protocol was also conducted. Both PTP assay protocols consistently showed that SHP2 was activated during K562 erythroid differentiation.