Jump to content

  • Log in with Facebook Log in with Twitter Log in with Windows Live Log In with Google      Sign In   
  • Create Account

Submit your paper to J Biol Methods today!
- - - - -

Protein precipitation method

  • Please log in to reply
2 replies to this topic

#1 Peakos



  • Members
  • Pip
  • 1 posts

Posted 03 October 2013 - 06:54 PM

I would like to have your opinion about the different methods for protein precipitation.
I usually use Acetone or Methanol precipitation for most of my projects which are usually quantitation by iTRAQ.
Different other methods exist with their advantages and disadvantages.

TCA/Acetone seems great but i have heard that it lead to some degradations and modifications.
TCA alone seems a good compromise.
The use of chloroform is quite hard in my lab because of hard safety procedures.

So i wanted your advices. According to your experience, what precipitation method is the best for a good yield in general cases.

Also i don't use any benzonase or sonication after lysis ( I'm worried that even on ice, sonication can lead to carbamylation due to the very high increase of temperature localy during cavitation process and i use 6M Urea).
So do you think Acetone remove efficiently DNA ? Usually i got a very huge and viscous pellet directly after adding ice cold acetone to the lysate (Probably DNA ?) that i remove with a tip, then i let the protein precipitate over night).

#2 dimensionsbio



  • Active Members
  • Pip
  • 16 posts

Posted 04 October 2013 - 12:37 PM

I have never compared precipitation methods to be able to comment on the advantages of one over the other.  I would routinely use TCA precipitation with a final acetone wash step to dry the sample, but these samples were simply used for SDS-PAGE and nothing else.  After lysis of of your cells, DNaseI should be added to the lysate.  After sufficient time for enzyme to work, the viscosity due to genomic DNA is no longer an issue.  I would hesitate to remove anything with a pipette tip as a significant percentage of your protein may be present in what you are removing.

#3 labtastic



  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 205 posts

Posted 11 November 2013 - 05:57 PM

I've only ppt'd protein with acetone/tca for SDS-PAGE samples when they are too dilute to load the typical 1-10ug of protein needed for coomassie staining in the 20ul or so that gel lanes allow.


For doing this though, I always prefer acetone ppt'n because it's one less step. 1 part sample to 4 parts cold acetone, incubate -20C for 30 min, spin at 16k x g at 4C for 10min, aspirate and dry. Solubilize pellet in 1x sds-page buffer. Boil sample at 95C for 5 min and you're good to go. Quantitative recovery as well.


I only use TCA ppt'n when volume is an issue or when sample has so much salt that the salt isn't soluble in 80% acetone. Add 1 part sample to 0.3 parts 100% (w/v) TCA. Spin at 4C for 10 min, aspirate supernatant, wash pellet with cold acetone, spin again at 4C for 10 min, aspirate acetone and dry pellet, resuspend pellet in 1x SDS sample buffer. Quantitative recovery and gel samples always look clean. 


I've spent many a time encouraging lab mates to take the time to do these ppt'ns when their protein samples are too dilute to run on a gel. Instead they just prefer to run max volume of their samples on the gel and squint for hours trying to interpret the most faintest of bands on gels and getting nothing out of it....all in an effort to save 45 min of time to get no results. 

Home - About - Terms of Service - Privacy - Contact Us

©1999-2013 Protocol Online, All rights reserved.