3M Dialysis Cassette Protocol 1. Prepare dialysis buffer a. Need enough buffer to do 3x exchanges at 50:1 buffer to sample ratio b.

Dialysis Buffer for crystallization i. 1% Beta-OG ii. 100mM NaCl iii. 10mM Tris-HCl pH 7.0 2. Hydrate cassette a. Be careful not to touch cassette membrane, as any contact could tear the membrane b. Fill a beaker with an appropriate amount of buffer (in this case ~150mL) c. Place a stir bar at the bottom d. Hydrate the membrane by placing the cassette into a float and setting it into the buffer for ~30s (1 min for larger cassettes) 3. Inject sample a. Use a 23 or 21 g. needle on a 1mL syringe b. Draw up sample, including a small pocket of air c. Inject into on of the four ports, being careful to go as little as possible into the actual chamber d. After injection, make sure all sample has been expended by pushing a small amount of air into the chamber e. With the needle still in the cassette, carefully invert the cassette so that the sample settles at the bottom of the chamber f. Draw back on the syringe to pull out as much air as possible, increasing the surface area of the sample WITHOUT damaging the membrane 4. Dialyze at 4C with gentle spinning a. Perform dialysis, keeping the cassette so that the port used for injection is pointing up away from the buffer b. First exchange : 4 hours c. Second exchange : 1 hour d. Third exchange : overnight 5. Remove sample a. Remove the sample carefully, using the same size needle and syringe b. Cassette can be discarded