Acetone washes for TCA precipitation protocoles - (Feb/22/2011 )

I have to concentrate the protein content of a Optiprep (iodixanol diluted in PBS 1x) velocity gradient.
I used 15% TCA for my precipitation and after 15 000 g centrifugation I could see white protein pellets in the right fractions.
The problem is, when, after removing the supernatant, I added acetone to wash the pellet, the solution became foggy and something precipitated (I assume it's the remaining Optiprep.) If I take care of neutralizing remaining TCA can I skip the acetone wash and resuspend my protein pellet directly into sample buffer ?

Or does anyone have another idea ?

Thanks,

-Louppy-

Louppy on Tue Feb 22 20:31:33 2011 said:

TCA assay if you want to run SDS-PAGE

To 200ul of sample add 200ul of 10% TCA.Leave on ice or at -20 for 20 mins.
spin for 10 mins.
Add 100ul of ice-cold ethanol disacard the supernatant. (Repeat this step twice).
Dry the eppendorf by placing it in the hot air oven for 2-3 mins. Once its dry add 10ul of 2X treatment buffer (u will notice brown color).
Boil the sample 5-7 mins and load the sample in the gel.
Hope it works.

-priyaa Madhukaran-

we used to neutralize residual tca in our samples by adding 0.5M tris, pH 9, 5ul at a time (sample was 65-75ul with sample buffer), until the bromphenol blue turned blue.

-mdfenko-

Thanks a lot,

I've tried to mix �thanol with Optiprep and it didn't precipitate. I'll try it for my precipitates washes to see if my problem is solved !

-Louppy-