9 replies to this topic

[milovent]

Hi, i have a question.
I did protein precipitation using TCA method. The protein sample is BSA 1mg/ml.
After I mix the sample with BSA, and centrifuge it, I can see a small pellet. However,
after I mix some cold acetone to wash it, the protein disappear! And this happens all the time.
I tried 10 times, and the same thing keep happening. Cold acetone is supposed to wash the remaining TCA, right?
But, why now it dissolves my protein pellet?
Thanks before for your help.

[FlicFlak]

You dnt need to use acetone, that will just soluabilise the protein again. after centrifugation, just aspirate off the excess TCA and leave the pellet. then just resuspend in whatever you want. I do this all the time and never get a problem, never need to use a wash step!!

[milovent]

Thanks for the reply. But, I just followed the protocol, which requires the washing step.
Acetone is not supposed to dissolve my protein, right? but why this happened? is my acetone contaminated?

[mdfenko]

acetone, especially if ice cold, will not dissolve your protein.

when the acetone removes residual tca the pellet will lose its opacity. you should treat the tube as though the pellet is apparent and confirm its presence.

[Dianarodriguez]

mdfenko, on 25 July 2011 - 08:35 AM, said:

acetone, especially if ice cold, will not dissolve your protein.

when the acetone removes residual tca the pellet will lose its opacity. you should treat the tube as though the pellet is apparent and confirm its presence.

i use normaly is cold etanol for the last step and work very well!  for example 30ul protein + 30 ul TCA 10% + 30ul watter let on ice or 4° 30min than centrifuge and wash with 60 ul of ice etanol.

may be you can me help with a quetion pleace :
i have a small protein with his-tag and tevsite. i have very nice expression but the problem is,that the protein no binding in to the Ni-NTA. the protein is in the soluble fraction. i trie many buffer and too witout imidazol but no work , i trie to in GdmCl  6M and no work, because for me is no importan if my amilin peptid 37 aa. is folding or no , but no work

[z.biochemistry]

hello.my research is about depletion albumin from human serum.I wanted to know tc.aceton is suitable for my work

[mdfenko]

z.biochemistry, on 26 September 2011 - 10:52 PM, said:

hello.my research is about depletion albumin from human serum.I wanted to know tca/acetone is suitable for my work

no. tca/acetone will precipitate all proteins. it is not selective.

[z.biochemistry]

but i read an article that removed albumin by TCA/aceton.
name of article:
A modified protein precipitation procedure for
efficient removal of albumin from serum(2005)

[mdfenko]

the abstract doesn't give any specific information on the procedure. i would guess that they try a fractionation by varying amounts of tca/acetone to attempt to selectively remove albumin and the other most abundant proteins.

[z.biochemistry]

sorry.are
there more polar solvents than water?