Cell lysates - Protein precipitation? - (Nov/16/2010 )

hi there. i have cell lysates that are in RIPA lysis buffer.
however, the concentration (After doing a BCA assay) is too low due to possibly adding too much buffer to my plate of cells.

i want 30ug of protein per well when i do my western blot. Unforutnately, with the concnetrations i have,this would require 40ul of sample, which doesnt fill the well.

Is there a way of precipitating the protein to make it more concentrated, that wont denature the proteins in doing so?

I check my proteins using a BCA assay, so id also need the protocol to not affect the BCA reagents used in this.

Thanks!

you can use a spin microconcentrator (amicon/millipore).

you can also precipitate with (cold) acetone or ethanol, various salts (especially ammonium sulfate) or polyethylene glycol.

mdfenko on Tue Nov 16 18:51:43 2010 said:

i used to do acetone precipitation. slowly add ice cold acetone to ice cold protein solution, with gentle mixing. centrifuge, aspirate supernate and resuspend pellet in desired medium to desired volume.