13 replies to this topic

[dr_fthiebault]

Hye

I found a protocol to purify protein with a mix of TCA, mercapto and aceton, do you know it and have you a backgournd with this process?

[Shubenok]

Purifing from salts, nonpolar contaminants or other proteins ?
Please write more, and we`ll try to help You.  

[keithwu]

I'm going to concentrate my protein sample by the TCA/acetone method too. The concentrated protein sample prepare for western blot or 2D gel.
So the % of protein recovery is my main concern. Also, I heard that the precipitated pellet is difficult to dissolve. Do you know the best way to re-dissolve the pellet?
I'll share my experience after my trial.
Anyone have experience and suggestion in such method?

Any suggestion is welcome, and many thanks!

[Shubenok]

I usually use acetone + 0,1 HCl (for better precipitation) and maximal cooling, and the only way to dissolve precipitated protein with good result  is to use urea (for example).
When using TCA You must know, that in some cases easy to obtain hydrolised products (big and small peptides), so after addition TCA,  add gaseous NH3 to neutralise solution.
In any case after such methods You will obtain denatured protein (certainly with exceptions).

Edited by Shubenok, 04 February 2004 - 05:57 AM.

[keithwu]

so TCA is too acidic and leading to protein modification...
and do you mean 0.1M HCl?

[Shubenok]

Yes, 0,1 M

[quol]

What is the mininal/optimal concentration of proteins for efficient precipitation with TCA? I lose my protein in the precipitation because of too low concentretion and I need to add some extra protein to the solution (I use BSA) but I don't want to overload the SDS gel.

[kawaka]

Hi Quol,

You may want to try precipitate your protein using TCA in acetone.

The combination of TCA and acetone is commonly used to precipitate proteins during sample preparation for 2-D electrophoresis and is more effective than either TCA or acetone alone. Suspend lysed or disrupted sample in 10% TCA in acetone with either 0.07% 2-mercaptoethanol or 20 mM DTT. Precipitate proteins for at least 45 minutes at ?0 ºC. Pellet proteins by centrifugation and wash pellet with cold acetone containing either 0.07% 2-mercaptoethanol or 20 mM DTT. Remove residual acetone by air drying or lyophilization.

Also please try this protocol
TCA-DOC
For precipitation of very low protein concentration
http://www.ls.huji.a...cipitation.html

Help it works for you.

Edited by kawaka, 18 June 2004 - 06:32 PM.

[phdconsult]

Plan on using tRNA at 1 mg/ml as a seeding agent. It will not interfere with PAGE analysis and will definitely aid in precipitation.

[Sprag]

I want to concentrate my samples after sucrose gradient using TCA/DOC (6.5% TCA, 0.05% Na deoxycholate), but the samples which contains Triton (from the lysis buffer) does not precipitate and become kind of viscous. The viscosity completely disolves when washing with 80% acetone. The samples, which doesn't contain Triton, precipitate nicely using TCA/DOC, so the procedure works, so I think the problem is the Triton.

Any suggestions or similar experience?  Cheers...

sprag

[phdconsult]

Try a 1:1 dilution of the recovered fraction using buffer containing no Triton

[aloks321]

Hello,

I am trying to isolate a protein who has a cysteine rersidue. I am testing if this cysteine is making a disulfide bond with another monomer as a mode of regulation. I found a paper who was saying that proetins redox state can be fixed by pption with TCA but it doe snot give a detailed protocol plus they work on E.c oli while i am working on bacillus. so anyone could help me what to do exactly? Thank you in advance.

ali

[serglom]

Anybody knows if TCA precipitation affect to protein phosphorilation?

Thanks a lot

[Richer]

serglom, on Jan 12 2005, 12:19 AM, said:

Anybody knows if TCA precipitation affect to protein phosphorilation?

Thanks a lot

seems not there is a EMBO Paper adressing this Delaunay A et al "H2O2 sensing through oxidation of the Yap1 transcription factor" see Meterials and methods!
does anyone did an IP after a TCA precipitation?