Knight:Dialysis

Purpose
To change buffers in which your protein resides.

Note that although this method should work, I find that I lose my protein using this approach. Try Knight:Centrifuge desalting instead.

Materials

 * Slide-A-Lyzer MINI Dialysis Unit from Pierce (video, manual, product page, applications guide)
 * In general, the molecular weight cutoff for your column should be about half the molecular weight of your protein of interest.
 * dialysate (i.e. destination buffer)

Procedure

 * 1) Wear gloves to prevent contamination.
 * 2) Optional: to remove contaminating glycerol from the dialysis unit, dialyze the unit in 1L of DI water for 15 mins. (Glycerol content: <3% in 3K, ~15% in 7K and ~23% in 10K MINI)
 * 3) Optional: to remove contaminating metals, dialyze 15 minutes against 1 L 1 mM EDTA. (Metals present in a 3K, 7K or 10K MINI; 2 ppb iron, 5 ppb magnesium, 1.5 ppb nickel, 0.2 ppb zinc, 0.2 ppb copper, 0.5 ppb chromium and 0.3 ppb cadmium)
 * 4) Apply sample with a standard pipette.
 * 5) *Sample volume should be between 10-100 &mu;L.
 * 6) Cap the dialysis unit and place in a floatation device.
 * 7) Add 0.5-1L of dialysate to beaker.
 * 8) Add stir bar.
 * 9) Place the beaker in ice or in a cold room and on a stir plate.
 * 10) Place the float with dialysis unit in the beaker so that the bottom of the dialysis unit is in contact with the dialysate.
 * 11) *The volume level of the sample should be higher than the dialysate to avoid hydrostatic pressure forcing dialysate into the unit (thereby diluting the sample).
 * 12) Use a low speed setting on the stir plate to avoid submerging the unit.
 * 13) Equilibrate for 2 hours.
 * 14) Collect the sample from the corner of the Slide-A-Lyzer unit.