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!
Photo
- - - - -

protein concentration


  • Please log in to reply
5 replies to this topic

#1 hsk

hsk

    member

  • Members
  • Pip
  • 2 posts
0
Neutral

Posted 26 September 2003 - 01:22 AM

Hi!
I have been trying to concentrate protein. Our lyophilizer showed the proper temp and vaccum settings but still my sample thawed. Any idea why? What other option do I have, as I want viable protein and my sample is in litres?

Thanks in advance

#2 jadefalcon

jadefalcon

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 223 posts
1
Neutral

Posted 01 October 2003 - 06:40 AM

Hi there!

If your protein is big enough I could recommend you the method used in our lab.

I use Amicon Ultrafiltration Stirred Cells for concentrating a 24kDa protein routinely.
The largest cell, to my knowledge, holds up to 400ml solution.
I'm concentrating up to 2L of protein solution successively in 5 turns, and it works fine for me.
I use filters with 3,500Da molecular weigth cutoff (MWCO), so nothing is lost. If your protein is smaller, you will have to try it yourself once. The manufacturer says it's safe for proteins larger than 2 times the theroretical MWCO of the filter mebrane, but that could be a bit close. In my case the 24kDa protein is filtered through a 10kDa MWCO membrane. The concentrated protein is native and functional in all assays.

And you can diafiltrate your protein at high speeds with much less buffer waste after concentration...

Mike
--- He who finds typos may keep them! ---

#3 hsk

hsk

    member

  • Members
  • Pip
  • 2 posts
0
Neutral

Posted 06 October 2003 - 07:10 PM

Hi!
Thanks a lot. Might try it.

#4 kenny

kenny

    member

  • Members
  • Pip
  • 2 posts
0
Neutral

Posted 06 April 2004 - 06:31 AM

you can also try vacuum centrifugation... have you heard about this?

#5 senmehmet

senmehmet

    member

  • Active Members
  • Pip
  • 7 posts
0
Neutral

Posted 06 April 2004 - 08:35 AM

My protein requires 20 liter buffer, but I used Ammonium sulfate to precipitate my protein into 20ml. Very beneficial, but a bit nasty. Almost all kind proteins will precipitate at 58% ammonium sulfate, and you can dissolve this pellet in 10 or 20ml of your buffer where you can store your protein. I know, you can dialyse your protein aganist PEG or sucrose. Primitively, I like concentrating peptides by steam. You put your peptide into dialysis, and hold it with clamp to holder. Then, start boiling water in a beaker under this dialysis bag, water of your peptide or protein will come out and evaporate. I do this under hood. WHile water get out of the dialysis, evaporation keep the dialsis bag enough so that you will not face temperature denaturation.
I hope, you can cope with your problem
Mehmet

#6 phdconsult

phdconsult

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 89 posts
0
Neutral

Posted 08 April 2004 - 01:15 PM

If a sample liquefies under lyophilizing conditions despite correct vacuum and temperature settings, electrolyte balance of the solution must be restored. This is done by adding water 15%v/v. Strange that it may sound -it works just fine. Possibly the addition of water reconfigures the entire solution. You will see no more thawing.

phdconsult




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

©1999-2013 Protocol Online, All rights reserved.