Matt Gethers/CRI, Thailand/Labwork/Dialysis

Dialysis

1. Get dialysis tubing and cut more than enough to hold the volume you intend to dialyze (2 ml/cm of tubing). Be sure the tubing has a pore size appropriate for the molecular weight of your protein.
2. Fill a beaker with RO water and wash the tubing. Be sure to separate the sides of the tube so the inside can be washed.
3. Use a clamp to seal one end of the tubing. Be sure the tubing is flat and is in the middle of the clamp.
4. Fill the tubing with your sample and clamp the open end.
5. Fill a beaker with 1 L of dialysis buffer (see contents below) and place the clamped tubing in the beaker.
6. Add 1 ml 1 M DTT to the liter of buffer.
7. Place the beaker at 4^oC on a stir plate over night.
8. When dialysis is complete, unclamp one end, pour the sample into a tube and place on ice.
9. To estimate the final concentration of the sample, divide the moles of salt both in the sample and the buffer by the total volume (1 L).

Reagent	Volume	Final Concentration
5 M NaCl	20 ml	50 mM
500 mM EDTA	4 ml	1 mM
1 M DTT (Don't add to master)		1 mM
1 M Tris pH 7	100 ml	50 mM
100% Glycerol	100 ml	5%
H20	Up to 2 Liters

Be sure to add 1 ml 1 M DTT to 1 Liter when ready for use.

Run Notes

7.28.08

I made 2 liters of buffer as per the protocol above. I'm not prepared to dialyze samples yet, though, so I've placed the 2 liters in the four degree. No DTT added.