The his-tagged protein was captured initially using an affinity column (Ni-NTA), which was followed by anionic exchange (Q resource) and finally gel filtration (Superdex 100) on a Pharmacia FPLC system.
The protein was purified from the media using affinity Ni-NTA chromatography followed by stabilization of purified protein in 50 mM Tris-HCl buffer at pH 7.4.
Recombinant proteins were purified to homogeneity using metal chelate affinity (Ni-NTA, Qiagen) chromatography and the His-tag cleaved with thrombin (Novagen).
Briefly, his-tagged syntaxin-1A, synaptobrevin-2, SNAP-25A, and synaptotagmin-1 were expressed in E. coli and purified using a combination of Ni-NTA affinity (Qiagen, Hilden, Germany) and size exclusion chromatography on a Superdex 200 column (GE Healthcare).
The His-tagged proteins were extracted from the supernatant of the cell lysates with the addition of 1 mmol/L protease inhibitor PMSF and 1 1000 cock-tail proteases (Sigma), purified by Ni2+-affinity chromatography using a 1 mL Ni-NTA column.
The total soluble protein was extracted and the 6 × His-TtsfGFP was purified using an Ni-NTA affinity column.
The proteins were purified using metal affinity beads, Ni-NTA (Ni2+ nitrilotriacetic acid) agarose resin (Qiagen, Venlo, The Netherlands).
To examine the properties of the lipase produced by E. coli BL21/pACYCDuet- lipA- lipB, the recombinant lipase was purified to homogeneity (more than 95%) using nickel affinity chromatography (Ni-NTA) by exploiting the histidine tag.
RNAP was purified from ORB4028 (spx::neo his10-rpoC) as previously described using a Ni-NTA affinity column, a heparin agarose, and a Bio-Rad High Q column [11], [32], [33].
Proteins were purified from the lysis supernatant using a Ni-NTA affinity resin (QIAGEN, Valencia, CA), eluted from the column in lysis buffer with 250 mM imidazole, and then dialyzed into lysis buffer to remove the imidazole and concentrate the purified protein.
