X-tal protocols: Equilibrium Dialysis with acrylamide plugs.

Equilibrium Dialysis with acrylamide plugs.

Acrylamide plug.
Diffusion controlled acrylamide plug.

Acrylamide plug.

Acrylamide plug

C
P
O
A - Capillary
- Protein
- Outer solution
- Acrylamide plug

Zeppezauer et al. [Zeppezauer, 1968] described the use of polyacrylamide, polymerized in capillaries, as a dialysis membrane for the crystallization of proteins. In this procedure capillaries are dipped in an acrylamide solution to obtain a liquid column of about 1 cm at the end of the capillaries. After polymerization the capillaries are filled with buffer and placed for 2-3 days in the start buffer to allow residual traces of acrylamide monomers and polymerization catalysts to diffuse out of the gel. Crystallization experiments are started by withdrawing the buffer with a thin tubing and the injecting the protein solution into the cell. The upper end of the capillary is closed with paraffin. The capillary is placed vertically into the outer solution standing on Pyrex-wool. If crystallization occurs only at high salt or solvent concentration, the polyacrylamide plug can shrink excessively and detach from the inside wall of the capillary. In such cases, the polyacrylamide plug should be prepared from a monomer solution containing the buffer and about half the concentration of salt required for crystallization.

Diffusion controlled acrylamide plug.

Acrylamide plug2

C
P
O
A - Capillary
- Protein
- Outer solution
- Acrylamide plug
The rate by which equilibration is reached is determined by

the length of the acrylamide plug.
the diameter of the capillary.
the volume of the protein solution.
ion concentration and mobility.
concentration of acrylamide.

To control the rate of dialysis, the height of the acrylamide plug can be varied by injecting the polymerizing acrylamide solution into the capillary.
The capillaries (Disposable micropipette, cut in half) were fixed with tape at an angle of about 10° (See picture). The acrylamide concentration has to be consistent with the size of the protein to prevent the crystal from growing too far into the polyacrylamide (used 15% for 89 kD protein). The acrylamide solution is prepared without the TEMED. A 100 ul aliquot was taken and TEMED (around 0.2 ul) was added to start the polymerization. The amount of TEMED should be enough to polymerize the solution in 40 sec. It is important that the acrylamide polymerizes quickly, because oxygen terminates the polymerization and the height of the polymerized part of the plug will vary too much.
The quickly polymerizing acrylamide solution was pipetted into the capillaries, around 10 capillaries were done before the solution polymerized. The pipetted volume determined the height of the plug. After polymerization the capillaries were placed in a tube filled with the start buffer above the top of the capillaries and spun to fill the capillaries completely with buffer. Equilibration with the start buffer was allowed for 2-3 days.
Crystallization experiments were started by removing the start buffer from the capillaries by placing the capillaries upside down in a tube with a tissue at the bottom followed by spinning in a hand centrifuge. The protein solution was injected into the capillaries and layered on the polyacrylamide plug using the hand-centrifuge.
The capillaries were placed in a 15 ml Falcon tube with 1 ml of the outer solution. [Zeelen, 1992]
Filling capillaries

Capillaries fixed with tape on a box, at an 10° angle, filled with acrylamide.


C P O A	- Capillary - Protein - Outer solution - Acrylamide plug