Protocol

Abbreviation
NU_tag-off_purification
Name
NU tag-off purification
Laboratory name
Northwestern University
Type
purification
Description
Proteins are purified by Ni-affinity chromatography on HiTrep FF and desalting HiPrep 20/10 columns using AKTA Express system. Pure proteins are collected in buffer, containing 10 mM Tris-HCl (pH 8.3, 500 mM NaCl and 5 mM b-mercaptoethanol. Samples are concentrated to 5-10mg/ml depending on solubility. For purification tag cleavage proteins are mixed with TEV protease in 1:20 ratio and dialysed (while been cleaved) with 10mM Tris-HCl (pH8.3), 500 mM NaCl, 1 mM EDTA, 5-10% Glyserol, 0.2-0.4 M Arginine, 5 mM b-mercaptoethanol for 1-2 hr at 37C and at least overnight at 4C or untill cleavage is complete (checked by SDS-PAGE). After complition of the tag cleavage proteins are dialysed with 10 mM Tris-HCl (8.3), 500 mM NaCl and 5 mM b-mercaptoethanol to remove EDTA, applied on Ni-HiTrap column and collected as a flow through. Samples are concentrated to 5-25 mg/ml depending on solubility and used for crystallization experiments. Unused protein is flash-frozen in liquid nitrogen and stored at -80C.