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 puriification using IMAC: flowthrough very slow


  • Please log in to reply
2 replies to this topic

#1 Bioscientist

Bioscientist

    member

  • Active Members
  • Pip
  • 26 posts
1
Neutral

Posted 15 March 2013 - 01:48 PM

Hi all

After extracting my protein in Tris-sucrose buffer, I do a dialysis to take out the sucrose. Then I centrifuge it to get rid of any precipitation. I then filter it using 45 um filters. Then add the Ni-NTA agarose to it and leave it overnight at 4 degree celcius. When I run it through the colum it goes very very slowly like 1ml in 1 hour.

So it basically takes me like ages to purify my protein.

Can anyone please suggest to me what I can do speed up the process? Other than using pumps as my lab don't have one!

Thanks.

Bioscientist

#2 Missle

Missle

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 132 posts
18
Good

Posted 18 March 2013 - 05:39 AM

Based on how you prepared your sample, I would say you have a problem with your column. Maybe your frit is clogged or is too small of a pore size or your drain port is clogged. Your flow rate is way too slow even for gravity flow and your sample shouldn't be causing that issue given the way you've prepared it.

Good luck!

#3 mdfenko

mdfenko

    an elder

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 2,803 posts
133
Excellent

Posted 18 March 2013 - 07:55 AM

since you appear to be using gravity flow, you can make the exit tubing longer and set the receiving vessel lower to increase the flow rate.

this principle is described in the gel filtration handbook available from ge healthcare.
talent does what it can
genius does what it must
i do what i get paid to do




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

©1999-2013 Protocol Online, All rights reserved.