Dear Amala,
1. Increase your buffer pH. As a rule of thumb, usually it is
recommended to work with pH 7.6 and above for Ni purification. Try pH 8.
2. Increase your buffer A [NaCl] to 500mM. Some proteins are more stable
in higher NaCl, and for many, 50mM is not enough. It is possible your
protein has already precipitated in the lower [NaCl]. You can also
increase glycerol to 10%.
Having said that, you didn't provide additional details (Protein's
origin - eukaryotic/ prokaryotic) - if it's from a eukaryotic source, it
could be your protein needs additional post translation modifications
(e.g. sugars etc) for stabilization, which most likely will require a
different expression system. If your protein is of a bacterial source, I
would try to add a solubility tag (eg MBP), change the location of the
His tag, or change expression conditions (lower temp, different strain).
There are plenty of other options, but I'd start with that.
Good luck,
Amit
On 03/01/2018, 05:56, amala mathimaran wrote:
I am trying to purify a protein using HIS-select Ni-affinity column
(washing with 50mM Tris pH7.5, 50mM NaCl, 30mM imidazole, 3mM BME,
5%glycerol and elution with the same buffer + 250mM imidazole, 500mM
Nacl). The protein (pI= 6.04) becomes cloudy/precipitated within
15minutes of after elution. I add EDTA to the eluted protein
immediately in the fraction tubes but the protein still precipitate,
can anyone suggest how to avoid precipitation of protein.
Thanks and Regards,
--
*********************************
Dr. Amit Meir
Professor Gabriel Waksman's group
Institute of Structural and Molecular Biology
Department of Biological Sciences
Birkbeck College
Malet Street
London WC1E 7HX
UK
