having hard time to re solubilize after protein aceton ppt - protein precipitation (May/07/2005 )

Hello,

Is there anyone who can give a help
with good pratical tips in resolublizing protein after acetone-TCA precipitation or Methanol.Acetone preciitation for 2D electrophoresis?

I am having real-hard time to resolubilize the precipitated proteins.
I tried 1-hour vortexing at RT or sonicated the pellete. But all of my tires were useless.

My first starting material was a cancer cell line.
and proteins were all soulble in the lysis buffer.
After lysis of the cells, I sonicated then ultracentrifuged to pellete out the debris.

Please tose me any type of advice.
All are appreciated very much.

Thanks alot.

hi

sure there isn't any salt in the sample? This could be the thing that is not soluble in the lysis buffer...(if its too much)?

I have problems in resolubilization when I dry my pellets too long in the SpeedVac.

Hi,
instead of precipitating to concentrate your sample, why don't you use centrifugation devices with membrane cutoff of different molecular weights?

You always lose some material, but there is no absolute technique where you don't. This works, and you can give it a try, if you are working with high volumes. There are also small volume devices with 3kDa cutoff.

If your sample in lysis buffer is a small volume you can try liophilization or speedvac at very low temperature, to evaporate the buffer.

Then you solubilize your sample in the smallest buffer volume you can to quantify previous to load on the 2D.

Good luck.

M

Hi Biokrag,
As Lordofthelittle have mentioned you should not dry to dryness your sample after acetone or any organic solvent concetration method, however, you still can use your ppt proteins for one dimension electrophorsis (i.e. SDS-PAGE) and not for 2D (i.e. IEF). If you want try first to check how your acetone ppt work, you can still boil your samples SDS-PAGE sample buffer for 2-5 minutes, centrifuge to peelt any unsloublized pellet (will be mostly salts) and then run on SDS-PAGE. I hope this helps, good luck!

Thank you very for your advice.

Hi Biokrag,
As Lordofthelittle have mentioned you should not dry to dryness your sample after acetone or any organic solvent concetration method, however, you still can use your ppt proteins for one dimension electrophorsis (i.e. SDS-PAGE) and not for 2D (i.e. IEF). If you want try first to check how your acetone ppt work, you can still boil your samples SDS-PAGE sample buffer for 2-5 minutes, centrifuge to peelt any unsloublized pellet (will be mostly salts) and then run on SDS-PAGE. I hope this helps, good luck!

Thank you for your advice.
Well I strated with UREA(4M) and 25mM(Tris-HCL, 8.0)

I am not sure how much salt were there either in the starting buffer or the cells.

But I got an info by you. Thank you.

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Thank you very much for your advice.
I will try.

best wishes.

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