1. Dilute (1:10) protein in 8 M urea or 6 M guanidine buffer into the cold refolding
buffer (50 mM Tris/1 mM EDTA/1M L-Arginine/1 mM Glutathione (reduced)/0.8 mM Gluthathione (oxidised))
by dripping the denatured protein into the refold buffer with constant
stirring at 4 °C.
1a. Altenatively dilute (1:10) denatured protein into the cold refolding
buffer (50 mM HEPES pH7.5, 0.2 M NaCl, 1 mM DTT, 1 M Non-Detergent Sulfobetaines
(e.g., NDSB256 or 1M NDSB201)).
2. Leave for 1 hour to overnight at 4 °C.
3. Concentrate the sample and dialysis against 50 mM Tris/1 mM EDTA.
4. Other redox pairs may be used in place of oxidised/reduced glutathione:
5. Other additives that may enhance native structure or inhibit aggregation:
-sucrose or glycerol (>10%)
-detegents: N-lauroylsarcosine, CHAPS, etc.