I am trying to purify protein which is His-tagged. I had cloned the gene in pET28a+ vector. Most of the protein is going in the pellet. But by trying to modify the growth condition and IPTG conc. I could get some amount of protein in supernatant which I can see by running the sup in SDS-PAGE 10%. Protein is binding to the beads (Ni2+-NTA) but it is not getting eluted with elution buffer. I am using Ni-NTA magnetic beads from Qiagen.
Extraction buffer: PBS, DTT, Triton X-20, lysozyme- sonication
washing buffer:
50mM Sodium phosphate pH8
0.3 M NaCl
20mM Imidazole
Elution Buffer:
50mM Sodium phosphate buffer pH8
0.3 M NaCl
250mM Imidazole
Hi there,
You may try higher imidazole concentration and/or elution with EDTA to strip nickel from the magnetic beads. If interaction is specific then your protein should be efficiently eluted with these harsh conditions. If your protein is aggregating on the beads then it should remain associated to the beads.
Just in small scale you could try to elute with buffer containing up to 8M Urea. At least you would figure out if you have an aggregation problem. DTT is usually not recommended on Ni-Beads. If you urgently need reducing conditions I would recommend to change to a TRIS buffer and add 0.5 mM TCEP, otherwise maybe just leave DTT away. Good luck
In addition to previous answers, your protein probably goes into inclusion bodies inside the bacteria. In these cases, you can solubilise the protein (before the spin) in urea or GdnHCl, spin away cell walls and the like, then capture the denatured protein on the Ni-column. You then renature the bound protein by slowly reducing the denaturant concentration. Because the protein molecules are isolated from each other on the beads, they cannot form aggregates (this has been dubbed "infinite dilution"). Once this has been done, elute the protein from the column.
See also Yang et al.: Highly efficient production of soluble proteins from insoluble inclusion bodies by a two-step-denaturing and refolding method, PloS one 6:7 (2011) e22981, http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0022981.
if your protein expressed into the pellet then try to purify the inclusion bodies and after the preparation of the purified IBs, dissolve it in your buffer with 1% SDS you can also try PBS buffer for solubilization. Pass this supernatant to the Ni-NTA column .
Use different conc. of imidazole for elution optimization. you can add 0.1% N-lauryl sarcosine in elution and washing buffers.
After elution you can refold your protein by extensive dialysis.
for more detail you can see the attachment
Article Effect of pH on the structure, function, and stability of hu...
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