I have been trying to elute a protein using native prep. The prt of my interest is being expressed pretty well but is not binding to the beads.Most of the protein is going off in the supernatant post centrifugation (after sonication). i tried increasing the NiNTA bead volume, increasing binding time and also reducing imidazole conc to 5mM in lysis buffer but even after that i see that the prt not binding to beads. Any suggestions to improve the yield please????
The tag might be buried/not available due to interactions with other residues within the protein. Try using low concentrations of urea or other Ni-NTA-compatible chaotrope in your binding buffer to see if you can get the protein to bind.
There are two factors that are generally to blame when proteins don't stick to nickel beads: one is incorrect buffer conditions and the other inaccessible His-tags.
As Shruti mentioned, Ni-NTA works best at high pH. The minimum pH at which His-tags bind to the beads is 7.6 but at pH 8 to 9 the binding is enhanced. At pH lower than 7.6 histidines start to become protonated and therefore won't bind to the positively charged nickel ions.
If the protein doesn't bind under the correct buffer conditions, then I would do as Ulrich suggested and re-clone the tag from one end to the other.
Finally, I would also suggest using a 10His-tag instead of a 6His-tag, as they have much higher affinity for the nickel beads and stick more tightly to them. Apart from having longer tags that are more likely to be accessible to the nickel beads, proteins with 10His-tags generally elute at around 200 to 300 mM imidazole, which has the added advantage of giving you cleaner protein. Most contaminating proteins that bind non-specifically to nickel beads will have eluted by the time a 10His-tagged protein elutes.
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