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Updated: May 25, 2025
GST-His purification: A Two-step Affinity Purification Protocol Yielding Full-length Purified Proteins
Published on: October 29, 2013
Protocol for purifying biologically active microtubule-severing protein UNC-45A from E.coli using GST-affinity and
Asumi Hoshino1, Nimisha Krishnan2, Mihir Shetty1
1Masonic Cancer Center and Department of Obstetrics, Gynecology and Women's Health, University of Minnesota, Minneapolis, MN 55455, USA.
None:
Recombinant microtubule (MT)-severing proteins are valuable for studying their mechanisms of action; however, purifying them in an active state is challenging. Here, we provide a protocol to obtain biologically active and highly pure recombinant GFP-UNC-45A, a novel ATP-independent MT-severing protein, from E. coli. We describe steps for using GST-affinity and spin columns and detail procedures for assessing the activity of GFP-UNC-45A with in vitro MTs along with GFP-katanin as a positive control. The purified proteins can be used for downstream applications to study their functions. For complete details on the use and execution of this protocol, please refer to Habicht et al.1.
