2. Make sure your protein is in inclusion body. Membrane-bound protein may also be insoluble without detergent, but can be solublized by including low concentration (<1%) of SDS or Triton X-100.

3. Attempts to avoid inclusion body include growing bacteria at different temperature and using different bacteria strains and culture media.

4. Inclusion bodies are frequently solublized with a buffer that contains 8 M urea or 6 M guanidine hydrochloride.

5. Refolding may be achieved by:

    -Quick dilution, e.g., dilution of the proteins in 8 M urea to 0.4 M urea.
    -Refold on column, e.g., changing buffer from high urea or guanidine hydrochloride to no urea or guanidine hydrochloride while protein is being purified by ion exchange column or affinity column.
    -Slow dialysis against a buffer without denaturant.
    -Refolding in a refolding buffer.