Concentrating the protein sample is the last, and sometimes nerve wrecking step prior to setting up crystallization trials. I've heard the outcries of desperation reverberate through labs and hallways when the final sample precipitated in the concentrator device after a week of purification work. Concentrating proteins to higher than 10 mg/ml is risky business but necessary to generate a supersaturated protein solution and coax it into the metastable region of its phase diagram.
Here's a way, I think, would avoid the concentration step altogether:
Employ a modified version of the Dilution Method (see here or here) that is based on this paper:
Dunlop, K.V. & Hazes, B. When Less is more: a more efficient vapour-diffusion protocol. Acta Cryst. (2003). D59, 1797-1800
In a traditional vapour diffusion crystallization experiment, equal volumes of protein and well solution are mixed (i.e. 1uL + 1 uL). In the subsequent equilibration period, the drop shrinks from 2 uL back to 1 uL due to the vapor transfer. This brings the protein back to its initial concentration but in a different physical chemistry environment. The hope is that this different environment has a lower solubility for the protein and hence crystal nucleation and growth ensues form this supersaturated solution.
Using the Modified Dilution Method one would dilute the precipitant solution by for example 5-fold with water and use it to set up a 1 uL + 1 uL hanging drop or sitting drop. Note that the protein would not be diluted as described in the paper. Let's say the concentration of the protein at hand is ca. 2 mg/ml in a low ion strength buffer (or approx. 4 OD worth of sample)
What happens during evaporation? The excess water in the drop evaporates and shrinks the drop from its initial 2 uL to ca. 0.2 uL (the precise final volume would of course also depend on the composition of the protein buffer, lower ionic strengths protein buffers would be preferred with this Modified Dilution Method). With this reduction in volume, the protein concentration increases from the initial 1 mg/ml in the combined drop to 10 mg/ml.
'Back-of the envelope' sketch for the Modified Dilution Method for vapor diffusion protein crystallization experiments to avoid excessive protein concentration. Due to the initial dilution of the reservoir solution by a factor of 5 (prior to setting up the drop), the drop volume is reduced by vapour transfer until the reservoir solution is in equilibrium with the drop. In this case the drop shrinks by a factor of 10 from 2 uL to 0.2 uL, hence concentrating the protein from 1 mg/ml to 10 mg/ml. Note the use of Wizard I ;)
Since the crystallization regime of such crystallizations is different from conventional vapor diffusion based crystallization, such a dilution scheme could also be applied as a low-cost and simple way to optimize crystal growth.
Accurate manual nanoliter crystallizations prepared with realtively low initial protein sample concentration.
Try to beat that!
Peter
P.S. - let me know if it works.