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Star Republic: Guide for Biologists

Protein refolding

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:

    -Oxidized/reduced DTT
    -Cystamine/Cysteamine
    -Cysteine/Cystine

5. Other additives that may enhance native structure or inhibit aggregation:

    -glycine
    -sucrose or glycerol (>10%)
    -detegents: N-lauroylsarcosine, CHAPS, etc.