Problems with dialysis
Protein precipitation during dialysis against phosphate or Tris buffers (PBS or TBS) can be often caused by:
- too low salt concentration (NaCl, KCl)
- too high protein concentration
- sudden pH changes
- pH of dialysis buffer close to PI of a protein
Therefore:
- use at least 1000 fold excess of dialysis buffer
- perform procedure at 4c(at least 3 hours, followed by a buffer change)
- use at least 350 mM salt in the dialysis buffer
- optional: 50 % glycerol
Precipitated proteins are very difficult to refold. If possible purify your protein again and apply other conditions for dialysis.
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