Protein Crystallography Course

Additives

Popular additives are:

• Glycerol, which may stop nucleation and may give you fewer, larger crystals, and has the advantage of doubling as a cryo-protectant. Try anything from 1% to 25% glycerol.
• Ethanol or dioxane, which have the effect of poisoning the crystals and stopping too much nucleation
• Divalent cations like magnesium
• A detergent such as beta-octyl-glucoside

Hampton has three Additive Screens and three Detergent Screens each with 24 additives

Molecular Dimensions have a Popular Detergents kit (6 detergents) and an Expanded Detergents kit (58 detergents)

Ulrich Gohlke's Home Page contains a description of the Properties of Detergents.

Finding an appropriate additive can be related to finding conditions in which your protein is monodispersed. Try using different additives to get monodispersed protein (dynamic light scattering), and then set up a screen with that additive in all conditions


Oil on the reservoir

Diffusion can be slowed down by adding a layer of oil on the top of the hanging drop well

The proportion of light silicone oil to heavy paraffin oil must be mixed in different proportions to get an appropriate rate of diffusion
A novel technique to control the rate of vapour diffusion, giving larger protein crystals Chayen, NE, J. Appl. Cryst. 1997, Vol.30, No.Pt2, pp.198-202


Seeding

Seeding is a way of manipulating your position in phase space

There three different methods of seeding

1. Streakseeding. Enrico Stura has a figure
2. Microseeding
. See Douglas Instrument's paper "Crystallization of a protein by microseeding after establishing its phase diagram"
3. Macroseeding. Enrico Stura has a figure

and four different ways of using seeding

1. Homogeneous seeding. The seeds are used to grow crystals of the same lattice and symmetry from an identical macromolecule
2. Heterogeneous seeding (Cross-Seeding). The seeds are used to grow crystals of the same lattice and symmetry from a different, but usually very similar macromolecule (such as a mutant)
3. Homogeneous Epitaxial-Seeding. The seeds are used to grow crystals of a different lattice and symmetry from an identical macromolecule
4. Heterogeneous Epitaxial Seeding. The seeds are used to grow crystals of a different lattice and symmetry from a different macromolecule e.g. the nucleation of protein crystals on cellulose fibre impurities in the drops

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A watched crystal never grows
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