I am purifying my protein under denaturing condition and washing the inclusion bodies before with washing buffer (50mM Tris, NaCl-500mM, Urea-2M, Triton X-100- 1%, pH-6.8) and Suspension buffer (50mM Tris, NaCl-500mM,pH-6.8) one by one 5 times.
Thereafter Ni-NTA column was equiliberated in buffer containing (8M Urea, tris, 50mM, 500mM NaCl). Inclusion bodies were dissolved in same buffer and loaded on column. The Protein is not binding with column fully as you see in picture. (1st Lane is marker 2nd is loaded inclusion bodies, 3rd flow through, 4th 10mM Imidazole, 5th 20mM Imidazole). When column is washed using 10mM imidazole containing buffer all protein fall out with contaminants. Any suggestions to make protein purification possible.
What is the pH of your equilibration/run buffer? - it should be around pH 8.0 to get good binding (I use HEPES).
Its 6.8 but i used 7.2 and 8.0 before that and no success in protein binding.
Are you using loose beads or a FPLC column? Anyhow you should try to incubate the solution for longer time with the beads to get better binding.
Apart from that: your inclusion body washing makes it almost pure already.. I would just run it over a size exclusion in 6M Urea or bind it to a simple fast flow ion exchange column (leave the NaCl out first) and you will get your last missing bit of purity.
If the His-tagged protein does not bind to the Ni-NTA, a possible reason is that the protein is highly aggregated. This can happen with inclusion bodies even when they are solubilized in 8M urea. A test for aggregation is to perform gel filtration chromatography to see if the protein elutes as a monomer or an aggregate. If the protein is aggregated, you might be able to disaggregate it with 8M guanidine HCl or guanidinium isothiocyanate.
Thanks for your suggestions. I would try them and let you know the results..
Could you please check the existence of 6X His-tag of your protein or vector first. Then, if you change buffer pH or add some detergent (0.1-1% TX-100), still get the same result, please try ion-exchange chromatography, you may get the perfect results. Good luck.
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