I am looking for a protocol to purify proteins from RIPA. I need to do iTRAQ afterwards, so excess urea is not a good idea. Please let me know if you have any suggestions. Thanks.
Are you looking to buffer exchange your proteins out of RIPA buffer just to get rid of the buffer? Zebra spin desalting columns should do the trick for a low volume, simple buffer change up with minimal sample dilution. Not sure if you will get rid of every single trace of detergent though...
Most often a few washes with acetone and then suspension in buffer. A lot of people have seen that need to add detergents and/or urea to help solubilization, then dilution to acceptable concentrations of those reagents.
Other protocols are out there for ethanol precipitation. Same issues solubilizing the pellet, but these pellets seem to be a little softer than acetone precipitated pellets.
Are your worried about the excess urea for it inhibiting a proteolytic digestion of your sample? I suppose if you had a pellet that was precipitated and suspended in a high concentration of urea in a low volume, then diluted out you could manage a good digestion.