Methods and Kits to Create Protein Substrate˜HECT-Ubiquitin Ligase Pairs

摘要

Methods and kits to use in the isolation and identification of crosslinked protein substrate ubiquitin ligase complexes are disclosed. More specifically the methods and kits disclosed herein describe the use of bifunctional thiol-and-amine crosslinkers to covalently bind an endogenous or exogenous HECT-ubiquitin ligase to a downstream protein substrate, preferably in a cell lysate.

主权项

1. A method for forming a crosslinked protein substrate˜HECT-ubiquitin ligase complex in a buffer solution, comprising:
providing a buffer solution; adding a thiol-and-amine crosslinker to the buffer solution to create a mixture; and reacting the mixture to create a crosslinked protein substrate˜HECT-ubiquitin ligase complex.