Hi Bahja, did you purify your His- tagged protein from inclusion bodies?

You can try changing the pH of the buffer. You can try to do the crystallography with high salt. And you can try doing the crystallography with a less concentrated protein so that you don't need as much salt to keep it in solution.

Hi Steingrimur,

No, it is a soluble protein.

I would suggest ultra pure sterile water of pH 7 would do the job nicely especially if you do not know your protein nature

Regarding the suggestion of Khamis, I would not recommend using unbuffered water because you will not be able to control the pH. Also, as previously stated, the concentrated protein precipitates unless the ionic strength is high.

I would suggest increasing concentration of NaCl in the buffer. 250 or 500 mM NaCl concentration is still fine for crystallization experiments.  As you indicated your protein needs to be happy and stable prior crystallization screening. Otherwise you will observe crystallization drops containing heavy precipitate  Another option would be adding 5 % glycerol, but I would start by increasing a salt concentration first.  Also, I would suggest using Hepes vs. TRIS. 

The identity of the salt is another parameter that can be varied. You don't have to use NaCl, which is mildly chaotropic.

You could try to crystallize you protein at current concentration. 5-10 mg/ml is a good starting point. If you would like to fiddle with the buffer condition, check the following manuscript for inspiration. This should get you going. One more thing, you are not indicating any reducing agent in your buffer. I would recommend 2mM DTT or TCEP.

Jancarik, J., Pufan, R., Hong, C., Kim, S. -H., Kim, R. Optimum solubility (OS) screening: an efficient method to optimize buffer conditions for homogeneity and crystallization of proteins. Acta. Crystal. Sec. D. D60: 1670-1673. (2004)

Adam did you read her need that she is trying to avoid precipitation, that why I suggested ultra pure sterile water with pH 7, and I am recently working with proteins and peptides so from my own experience I only was able to keep protein stable as I mentioned and when I used Tris buffer pH8 I found that the purified protein has lost its stability

Thank you all for your help and suggestions.

Anton, screening buffers using thermoflour is absolutely worth doing and I would definitely do it for my proteins next time. 

Boguslaw, I used MES buffer instead of Tris and I reduced the pH to 5.5- it helped with solubility a bit but not completely. My protein definitely needs more salt! I did not try Hepes though. As for including a reducing agent in the buffer- I think it would disrupt disulfide bonds (if any) which is not suitable for crystallography.  Thanks a lot for the manuscript- definitely worth a read.

Khamis, I cannot use pH 7 since my protein has a pI very close to 7; so my protein would precipitate at this pH.

Bahjan, believe me you want to add reducing agent in order to prevent  the oxidation of free sulfhydryl residues (cysteines) in the protein which can lead to non-specific aggregation of the sample, sample heterogeneity etc.  Anyway good luck.

Sorry Bahja I did not notice that you mentioned the pI of your protein, however that could be solved by adjusting the water pH. Nevertheless its your choice to go whatever way works with you better