3 replies to this topic

[cm13]

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!

[mdfenko]

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.

[cm13]

mdfenko, on 16 November 2010 - 10:51 AM, said:

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.

I havent got a microconcentrator.
Would anyone have a protocol for ethanol precipitation?

[mdfenko]

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.

Edited by mdfenko, 19 November 2010 - 12:40 PM.