Hexa-His

Hexa-His (6X His Tag) is a commonly used affinity tag made up of six histidine residues. HEXA-HIS can bind to affinity chromatography media containing transition metal ions like nickel (Ni²⁺) or cobalt (Co²⁺), making it useful for protein purification^[1][2]. In Vitro: Hexa-His for Protein Purification and Protein Analysis^[2]
(1) Construct a plasmid containing the target gene and Hexa-His tag, and introduce it into E. coli.
(2) Select positive clones and grow them in LB medium until the OD600 is about 0.6-0.8, then add IPTG (HY-15921, isopropyl β-D-thiogalactoside) as an inducer and continue to incubate for a few hours to induce the expression of the target protein.
(3) Collect the bacterial cells, resuspend them in lysis buffer, and sonicate to lyse the cells, then centrifuge to remove debris and obtain the supernatant.
(4) Prepare the Ni-NTA affinity chromatography column and slowly pass the lysate supernatant through the Ni-NTA column to bind the CTB protein with the Hexa-His tag to the column.
(5) After protein adsorption, wash the resin with 10 column volumes of 60 mM imidazole binding buffer. Elute the target gene with the same solution containing 1 M imidazole using 5 column volumes. Collect the protein and analyze it by 15% SDS-PAGE.