what is the best method to concentrate protein - (Aug/06/2006 )
i do lysis in 100ul and get 1ug/ul of the protein.I want to scale down the volume, so I add actone to precipiate it. while i try to redisolve it in RIPA buffer it doesnt dissolve completly, please can you advice on some other way to concentrate this protein solution...
Are you sure your trouble stems from your method of precipitation, and not from trying to dissolve your precipitated sample in too small a volume of RIPA buffer?
Have you tried heating the sample, say at 37°C, fo a while?
There are other methods of precipitating proteins here.
Homebrew's right Kathy, might not be the precipitation step.
Other techs you could try though are dialysis, polyethylene glycol precipitation or the humble centrifugal concentrator (essentially, you spin your sample in a tube that contains a membrane filter...water goes through but larger molecules (your protein) are retained. Just don't spin too long or you'll end up with dry protein.
We usually run a gel after each stage in the process to confirm we have protein and give an indication of yield at each step.