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14. Remove 250 ml buffer/guanidine and add 250 ml buffer without guanidine. Continue dialysis >2 hr. Repeat.

With some proteins, renaturation by dialysis may require longer dialysis periods and more gradual decrements in the guanidine concentration of the buffer. Conditions for each protein must be determined empirically.

15. Remove dialysis bag to a container containing 500 ml of fresh buffer without guanidine at 4°C. Continue dialysis 2 hr to overnight.

16. Remove sample from dialysis bag, divide into aliquots, and freeze at -70°C or in liquid nitrogen.

If protein precipitates during dialysis, solid-phase renaturation (see Alternate Protocol 2), in which protein bound to the column is renatured before elution, should be employed.

17. Analyze fractions and process protein (see Support Protocol 1).

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