Knight:Dialysis

From OpenWetWare

Revision as of 15:41, 5 October 2006 by Reshma P. Shetty (Talk | contribs)
(diff) ←Older revision | Current revision (diff) | Newer revision→ (diff)
Jump to: navigation, search

Contents

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.
    • Sample volume should be between 10-100 μL.
  5. Cap the dialysis unit and place in a floatation device.
  6. Add 0.5-1L of dialysate to beaker.
  7. Add stir bar.
  8. Place the beaker in ice or in a cold room and on a stir plate.
  9. Place the float with dialysis unit in the beaker so that the bottom of the dialysis unit is in contact with the dialysate.
    • 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).
  10. Use a low speed setting on the stir plate to avoid submerging the unit.
  11. Equilibrate for 2 hours.
  12. Collect the sample from the corner of the Slide-A-Lyzer unit.

Notes

Personal tools