strange problem with His purification - (Aug/02/2010 )
A strange problem is encountered during His Tag purification and I would greatly appreciate opinions from everyone.
The protein of interest appears to cause the Ni-NTA beads (Qiagen) to clump and become yellowish upon its addition. In addition, the efficiency is significantly lower with most of the protein of interest remaining in the first eluant. The buffer condition, PBS with 10mM maltose. The NiNTA beads were pre-equilibrated with the same buffer. Initial attempts to rectify this situation included changing to Tris buffer without success. Denaturing condition was also used but no improvement was observed as well.
Is it possible that the protein of interest is incompatible with the Ni-NTA beads? If so, what reason may it be? In addition, what other factors should I consider for troubleshooting?
Thank you for reading!
Please let me know complete composition of buffer and is there is DTT, present in your buffer ?
( Which you re using for binding.)
With Thanks and Regards,