Purifying an ERM protein: is Ni-NTA compatable w/ 2M NaI?

Jonathan Kurtis, MD/PhD jkurtis at mail.med.upenn.edu
Wed Mar 31 22:33:43 EST 1999


Dear protein purification gurus,

I'm purifying an actin-binding protein from Pichia Pastoris (an
Ezrin-Radixin-Moesin family member). My 6xHis tagged protein of interest
(~80 kDa) co-purifies on NiNTA resin with an ~50 kDa band in a ratio of
85% protein of interest to 15% unwanted contaminant. This contaminant is
not removed by subsequent anion exchange or HIC chromatography despite
playing with detergents and ETOH and glycerol. It turns out that the 50
kDa contaminant is probably a binding protein (EBP50) that associates
strongly with ERM family members. In the literature, these two interacting
proteins have been separated by an affinity matrix (immobilized ERM used
to purify recombinant EBP50) with a 2M NaI elution.

My question: Can I bind my yeast lysate onto Ni-NTA in the presence of 2M
NaI?? This should allow only my protein of interest to bind with the EBP50
contained in the flow-through. Has anyone tried this or similar high salt
concentrations?? Anyone know what 2M NaI does to a protein??

Thanks for you help,
jake

-- 
Jonathan Kurtis, M.D./Ph.D.
-------------------------------
Department of Pathology and Laboratory Medicine
6th Floor, Founders
Hospital of the University of Pennsylvania
3400 Spruce Street
Philadelphia, PA  19104-6523




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