Typically TCA is applied to the sample first. Acetone is used to wash the precipitate. My simple protocol for this (also for removal of non-SDS-PAGE compatible substances like Guanidinium) is:

put fraction in vial

add 4 vol. 10% TCA

incubate 20 min on water/ice

spin at 20.000 x g for 15 min at 4 °C -> discard supernatant

add 1 vol ice-cold acetone to pellet

spin at 20.000 x g for 5 min at 4 °C -> discard supernatant

add 1 vol ice-cold acetone to pellet

spin at 20.000 x g for 5 min at 4 °C -> discard supernatant

dry for 10 min with open lid (or with preferably for 1 min with nitrogen gas to prevent oxidation)

dissolve pellet in 1 vol PBS / 5% SDS (here you can use less for concentration)

In some cases proteins do not precipitate for whatever reason. I would then try acetone precipitation (at -20 °C with 5 vol, 3 times over).

At first glance it looks to be backwards. You can concentrate proteins by adding TCA to a final concentration of 10% (I usually add an equal volume of a 20% stock), incubate on ice for 30 mins, then centrifuge at full speed in a benchtop 'fuge to pellet the precipitated protein. This is usually then visaile as a white pellet, although if there really isn't very much there then it might be hard to see. Carefully decant the liquid and wash the pellet with acetone (again, vortex and spin in the benchtop). Decant again and air dry the pellet (can put in a heat block at 50 degrees for 5 minutes if you are inpatient), before dissolving it in something like 8M Urea/2% SDS (obviously a smaller volume than you started with to concentrate the protein). Sample should then be good to go for your PAGE gel and Western blot.

Dear Lars and Rob

thanks for the insight.. Probably the protoclo reaaly have the acetone step misplaced...

I will try your suggestions, thank you very much.

70% ethanol, 100m TRIS pH around 8, for the washing steps after TCA is perfect for the begginers especially, since TRIS is buffering the acid, acetone does not.

Best - Jan

I can suggest removing the acetone from the first step and just keeping the TCA. And after the centrifugation, washing the pellet 4-6 times with acetone, then drying off the acetone 5-10 minutes on a 95 degree heat block then putting SDS-PAGE Buffer and resuspending and boiling the pellet, giving a final spin at full speed in a microfuge for 1 minute and loading the supernatant. That should help.

Depending on your starting volume and the molecular weight of the protein of interest, you can try vivaspin columns (several kDa cut-offs are available). This will allow you to reduce the volume of the sample without using harsh chemicals and should also aid recovery. I am assuming that the proteins are in a suitable buffer.