Hi,
although most widely used, Tris-NaCl buffers may be acceptable for many
but unfortunately by far not all proteins.
The solubilty of a protein can be modulated by the use of the proper
anions and cations. This all depends on the pH of the solution and the
pI of the protein.
Consult the following papers:
- https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1978149/ Crystallization
Optimum Solubility Screening
- https://www.ncbi.nlm.nih.gov/pubmed/15333951 Optimum solubility (OS)
screening
-
https://www.researchgate.net/publication/11105024_Importance_of_the_nature_of_anions_in_lysozyme_crystallisation_correlated_with_protein_net_charge_variation
The Hofmeister series plays a most important role!
Jeroen
On Sat, Dec 24, 2016 at 2:52 AM, Praveen Tripathi
<[email protected] <mailto:[email protected]>>
wrote:
Dear all,
I am graduate student working on a functional protein which i have
cloned in pET-28a vector for recombinant protein production in
E.coli expression system.
The expressed protein is purified on Ni-NTA resins with Imidazole
gradient. Surprisingly, i am getting distinct visible white
precipitate in pure fractions in eluted fractions itself.
Please suggest how to make it soluble or how to prevent the
precipitation.
On concentrator the precipitate ration is very much increasing. The
protein is pure in soluble as well as precipitate.
Buffer condition- 50mM Tris(7.5), 500mM NaCl, 10% Glycerol. Elution
buffer has varying concentration of imidazole varying from 10mM to
300mM.
Any kind of suggestion will be highly appreciated.
My project requires structure determination.
Thanks in advance.
Regards
Praveen
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