I'm working on isolating recombinant protein in E.coli (Ni-NTA)
I'am trying to purify recombinant protein that I overexpressed E.coli BL21(DE3)pLysS using the pCold1 vector.
The proteins have 6xhis on their N-terminus
The experimental conditions are as follows:
- E.coli strain : BL21(DE3)pLysS, vector : pCold1
- Grown in 500mL LB medium up to OD of 0.5
- Induction by 1mM IPTG at 37℃ for 6 hour
- Binding buffer : 50mM Tris, 200mM NaCl, 5% glycerol, 25mM imidazole, 1mM DTT, pH8.0
I elute with elution buffer using linear gradient ( 50 to 500mM imidazole)
Lane 1 : 225mM imidazole, Lane 2 : 250mM imidazole ...... Lane 9 : 425mM imidazole
But, even when the imidazole concentration is very high (over 300mM,), all the protein does not elute in the column.
it looks like histidine-tag aggregates.
Has anyone had any similar experiences ? I would appreciate any advice you may have. I'm so confused!!