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Refold protein 8M Urea: Sanjiv, The recommendations for rapid dilution and trying 6M GdmCl are great recommendations. As an alternative, I had a similar problem years ago with a membrane-associated receptor domain that was mostly found in inclusion bodies after expression (I assume this is your problem). We tried expressing at lower temperatures and in the presence of sorbitol, which are common methods for getting proteins to express in solution, but I found an alternative method worked. This will work if you have a tag for column purification (ie his tag). I incubated my pelets in 8M urea for a couple of hrs at 37C, spun and collected the supernatant, and then refolded the protein over a Ni column, as I had a his tag protein. To do this, you load and wash the protein normally, but before eluting, run b-cyclodextrin (i think 0.1%, you'll have to try a few concentrations) and then a detergent (I used Triton X-100). Then you can elute. Here's the paper I used as a guide: Biotechnol Appl Biochem 2006, 43:137-145 Hope that helps! Geoff -- Geoffrey K. Feld Krantz Lab College of Chemistry 492 Stanley Hall University of California, Berkeley "Vigilia pretium libertatis"