Have you tried simply using 2 volumes os 100 % ice cold acetone and leaving the utbe for 2-4 hours in a -20 freezer INSTEAD of using the precipitation kit? I favour acetone ppt because it´s simple and if you leave the tube overnight in a -20 you get a quantitative ppt of all proteins in the solution.

That´s my suggestion for sample prep of new samples, unfortunately about RECOVERING the proteins from the oily phase, sorry, no clue. :)

You can try dissolving the oily pellet in your Tris/SDS buffer and repeating the TCA precipitation.

Try to add to the mixture with acetone some ether till two phases resulted - super and precipitate.

Do you get this oily pellet before the acetone/water washes or after? I've never used this method, but the oily appearance of the pellet suggests lipid contamination to me. It may be that in the presence of high salt, some extracted lipid co-precipitates, and cannot be removed by a mostly aqueous wash.

I would try washing with 100% ethanol or acetone.

Wes,

up to centrifugation step for pelleting precipitate everything looks fine, after spinning samples the pellet is oily (before acetone wash).

I tried already acetone precipitation (however not O/N, I will try such a long precipitation), and methanol/chlorophorm method as well. I did not get precipitate that is large enough to recover it. My sampes are quiter dilute and for different precipitation methods I have to use large tubes that are not clear enough to see small pellets and have narrow entrance so not easy to operate with pipette.

Marcin,

Thanks for the information. Acetone precipitation may or may not work for you, but acetone or ethanol *washing* of your pellet may still be able to remove whatever contaminants are causing the oily appearance of your pellet.

A good collection of precipitation protocols can be found here:

http://wolfson.huji.ac.il/purification/Protocols/ProteinPrecipitation.html

If you know th pI of your protein try pptit with saturated ammonium sulfate

Mahmoud,

I have protein mixture so cannot tell pI.

Hello,

If nothing works you might try phenol extraction followed by acetone precipitation and washing the precipitate with 80% acetone.

I did dialysis and then precipitation - that was working well.

Thanks to you all!

Hi Marcin,

I get the same problem with an oily precipitate instead of a white fluffy one. Could you let me know your dialysis protocol?

I dialyzed against PBS with protease inhibitor coctail (in first incubation only). Five changes of PBS (10-volumes of sample) for 1h each at 4 deg. C. My samples were up to 5 ml so I used D-Tube Dialyzers (Novagen).

Thanks! I will try that.