Equilibrium Dialysis with Dialysis Tubes.

Micro-diffusion Cells.

C P O D T	- Capillary - Protein - Outer solution - Dialysis membrane - Tubing with feet

Micro-diffusion cells were first described by Zeppezauer et al. [Zeppezauer, 1968]: The microdiffusion cells, which may contain down to 10 microliters of protein, are made from 30 mm or 50 mm long capillary tubes of Pyrex glass or Plexiglas. The end of the tubes should have a smooth surface to avoid damage to the membrane. The dialysis membrane is attached to the bottom of the cell with a piece of soft, transparent PVC tubing. The tubing has two feet to hold the membrane off the bottom. The protein solution is injected into the cell, air bubbles must be avoided, and a continuous column of liquid in contact with the membrane should result. The upper end of the cell is closed with paraffin foil attached with a small ring of PVC tubing. this cell is placed vertically into a suitable vessel (50 ml Falcon tube) containing enough liquid to submerge the membrane. The final crystallization condition is reached by slowly changing the outer solution.

Hockey-stick.

H P O D T	- Hockey-stick - Protein - Outer solution - Dialysis membrane - Tubing

The hockey-stick [Kühlbrandt, 1992] consists of a glass capillary, bent by 90° near one end. The dialysis membrane is fixed with a ring of silicone tubing at the bend end. The protein solution is injected from the open end with a Hamilton syringe or a Gilson pipette, and shaken down against the dialysis membrane to remove air bubbles. The 90° bend helps to keep the dialysis membrane clear of sedimenting precipitate, that tends to block the pores when straight tubes are used. The tubes are easy to make by bending a longer tube and then cutting it with a glass saw. The cut ends are melted into a smooth surface which form a tight seal with a dialysis membrane.

Concentration-Gradient Equilibrium Dialysis.

C P O D T	- Capillary - Protein - Outer solution - Dialysis membrane - Tubing

A dialysis membrane is placed between an upper capillary which contains the protein solution and a lower capillary which serves as a connector to the outer solution. The lower capillary is first filled with the same buffer as used for the protein. The rate of dialysis is controlled by varying the lenght or diameter of the lower capillary.[Weber, 1970]