Gel techniques and small angle X-ray scattering to follow protein crystal growth

Agarose gels have been used in combination with small angle X-ray scattering (SAXS) at the synchrotron radiation facility LURE (Orsay) to follow hen egg white (HEW) lysozyme nucleation and growth. The advantage of using gels rather than solutions is that the nucleation centers, being trapped in the gel network, remain in the X-ray beam during the exposure. The advantage of agarose is that the scattering of the gel alone is limited to the low angle part. Nucleation of lysozyme was induced by decreasing temperature leading to supersaturation in the gel. The effect of varying different parameters was investigated: the percentage of gel and the concentration of salt, the effect of irradiation on crystal growth and the protective effect of an anti-oxidative agent. The crystals formed within one day were good enough to give Bragg reflections at small angles, easily observable with the camera at LURE.