I am attempting to purify a 34kDa protein that makes lots of inclusion bodies in E. coli. While isolating inclusion bodies, I noticed some of the protein also comes out in lysis buffer ( 10mM Tris-HCl, 5mM EDTA, pH 8.0) upon sonication.
I was thinking if I could purify protein from lysis supernatant using Ni-NTA. Can addition of MgCl2 say 10-20mM into this supernatant neutralize EDTA in the sample ? Or is it necessary to perform dialysis/ultrafiltration to remove EDTA?
If you insist of using Ni-NTA you have to remove the EDTA in before, even high quality Ni-NTA beads have an EDTA tollerance of around 1mM. Everthing above that strips the Nickel Ions of the beads.
However there is another, way easier option.
Use Ni-INDIGO beads instead. The INDIGO ligands bind the Nickel Ions 20 times more effective, so up to 20 mM. Then you do not have to include any new steps into your protocol.
More Information can be found here:
https://cube-biotech.com/products/protein-purification-products/his-affinity-resins-magbeads/indigo-ni/
You may use divalent cations to complexation of EDTA by decreasing the solubility. Primarily, buffer exchange using desalting robust way to implement.
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