I want to separate polysomic and non-polysomic proteins by sucrose fractionation, and then compare the fractions. If I precipitate the non-polysomic fraction by TCA standards directly from the sucrose (80%), all the sucrose precipitates together with the protein. How can I wash out the sucrose?
Hi Lena, I perform sucrose fractionation in order to isolate lipid raft domains. After collection of sucrose fractions, I succesfully apply a protocol for precipitation of very low protein concentration based in TCA and sodium deoxycolate (DOC). First, I add 2% DOC, vortex and let sit for 30min at 4ºC. Subsequently, add 1/10 (v/v) of TCA, vortex and let sit overnight at 4ºC. Spin 15min in microfuge at maximum speed at 4ºC, discharge supernatant and dry air the pellet. As optional, wash pellet twice with one volume of cold acetone. Good luck!
Dear Lena,
Ammonium sulphate is the technique I suggest. All the details are here:
https://www.researchgate.net/post/Can_anyone_suggest_a_Ammonium_sulphate_cut_protocol
Keep fingers cross!
Hi Lena
I agree with Santiago concerning the cold acetone.
it will wash your TCA pellet, to remove TCA and keep protein precipitation.
To preciptiate proteins within a sucrose-containing fraction I use methanol/chloroform precipitation. Then no sucrose will co-precipitate with the proteins.
150µL fraction + 600µL methanol. MIX. Add 150µL chloroform. VORTEX. Add 450µL water. VORTEX. Spinn maxspeed (eg. 14000xg) 5min. A white disc of proteins is formed between the organic and the aqueous phases - remove upper phase. Add 650µL methanol. INVERT tubes 3 times. Spinn maxspeed 5min. Remove solvent. Protein precipitate as a pellet.
My reference: http://www.mitosciences.com/PDF/sg.pdf
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