Hi Smile,

- you can try to increase the solubility of your POI by addition of certain stabilizing ingredients to your POIs buffer: A simple method for improving protein solubility and long-term stability. (Golovanov 2004)

- if you can manage to purify your POI soluble in first place, check the components of the buffer of your purified protein, find out what makes your protein happy and include that into your screening.

- or by cloning your POI to a fusion protein: A fusion protein system for the recombinant production of short disulfide bond rich cystine knot peptides using barnase as a purification handle. (HU Schmoldt 2005)

Soluble Regards

If your protein is precipitating at 1 mg/mL in crystallisation, you really need to work on the protein sample.

The first thing to try would be to try and find a stabilising buffer or compound. Differential scanning fluorimetry is a great method to do this. I've attached a good reference, although several companies now sell pre-made screens that are very convenient. That solved a similar problem for me in the past.

Otherwise, you should look at your protein, and see whether there are any destabilising elements present in the construct. Sometimes removing an unstructured element that is not relevant to your research question will improve solubility (and crystallisability). Of course, this is entirely dependent on your POI. It's always worth spending time thinking about whether your construct is ideal to give yourself the best chance of successful crystallisation.

Thomas' suggestion of using a very soluble protein to help with folding and purification is also worth considering.

Good luck!

You are not providing us with enough information to help you, i.e. the buffer conditions you use, or whether your protein is tagged, or untagged. The fact that you see instant precipitation at such low protein concentration would indeed indicate that you need to concentrate on the pre-screening part of the job. Your options are: 1) buffer screening to improve solubility of your POI, 2) Fusing your POI to a solubility tag and keeping it on for the crystal trials, 3) further reducing the crystallisation temperature, 4) crystallisation with a binding partner - this often contributes a lot of stability, 5) trimming your construct to get rid of flexible regions, which almost always hinders crystallisation.

Best of luck!

I have experienced the same problem for one of my proteins which was precipitated at even after purification at 4 degree.

I have found that excessive precipitation is due to the presence imidazole, to overcome this I have diluted the eluted fractions and dialyzed immediately overnight and precipitation was reduced to some extent.

I think following lines may also be  helpful...

1.You can check the pI of your protein and whether it is nearer to your buffer pH.

2.Try to increase or reduce the salt concentration (some proteins may show stability even with 50mM salt).

3.Check the % of cysteines and how much reducing agent (DTT/BME) you are adding to the buffer.

4.If your protein need a metal ion for activity/stability add low metal conc. in all purification buffers.

5.Some times leaching of nickel from the column may promote aggregation of the proteins, addition of small amount of EDTA may overcome this.

Good luck...

Thanks for everyone! I construct a new clone ,to delet some amino acides and the protein is happy  ,but it still not work when crystallization.