Help with Protein Precipitation
mark.bowen at stanford.edu
Mon Jun 25 06:18:41 EST 2001
You never mention what buffer you are using to solublize the protein. If it is
a membrane protein you must have detergent in sufficient quantities to protect
the hydrophobic segments. Usually concentrations above CMC are required. This
is very crucial as membrane proteins are highly variable in their solubility
requirements. Some membrane proteins absolutely require some amount of lipid to
Have you tried extracting your cells with high detergent and high salt (e.g. 5%
TX100 1M NaCl) or some other method rather than denaturants? This is better if
you can do it. It may require lowering the induction levels or decreasing
temperature with an longer induction time.
You can try small scale experiments for both extraction and solubility.
Lastly, in my experience, once membrane proteins start to aggregate it is very
hard to retreive them. They seem to form dehydrated beta structures that resist
solublization. This can result in "micro aggregates" that are very hard to
remove by precipitation.
More information about the Methods