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
- - - - -

Problem with Pierce GST spin purification column


  • Please log in to reply
6 replies to this topic

#1 biolcrazy

biolcrazy

    member

  • Active Members
  • Pip
  • 16 posts
0
Neutral

Posted 11 June 2011 - 10:00 AM

I am using the Pierce GST spin purification column for purifying my GST tagged protein. I have tried using the B-PER reagent/ sonication for bacterial cell lysis. I find that my protein goes in through the flow through fraction and I can't get any protein in the elution fraction. I incubated the cell lysate in the spin column at 4C in an end-to-end rotator for half an hour before spinning it around 1000 rpm to remove the unbound fraction. I add protease inhibitors and DTT to the cell lysate. I suspect that some of the glutathione beads are actually getting removed in the flow through fraction. Is it better to use Glutathione Sepharose beads instead of the GST- spin purification column?

#2 sify

sify

    member

  • Members
  • Pip
  • 2 posts
0
Neutral

Posted 23 June 2011 - 12:29 AM

Yes even I use sepharose beads for purification of my protein. Although its time taking process. But protein loss in flow through is very less.

#3 biolcrazy

biolcrazy

    member

  • Active Members
  • Pip
  • 16 posts
0
Neutral

Posted 07 July 2011 - 01:14 PM

Yes even I use sepharose beads for purification of my protein. Although its time taking process. But protein loss in flow through is very less.



thanks!
can you tell me which company's sepharose beads do you use?

#4 mdfenko

mdfenko

    an elder

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 2,709 posts
123
Excellent

Posted 15 July 2011 - 07:09 AM

sepharose is the brand name of the agarose from ge healthcare (nee pharmacia).

Edited by mdfenko, 15 July 2011 - 07:10 AM.

talent does what it can
genius does what it must
i do what i get paid to do

#5 qzlabs

qzlabs

    member

  • Active Members
  • Pip
  • 12 posts
0
Neutral

Posted 15 July 2011 - 03:09 PM

I am using the Pierce GST spin purification column for purifying my GST tagged protein. I have tried using the B-PER reagent/ sonication for bacterial cell lysis. I find that my protein goes in through the flow through fraction and I can't get any protein in the elution fraction. I incubated the cell lysate in the spin column at 4C in an end-to-end rotator for half an hour before spinning it around 1000 rpm to remove the unbound fraction. I add protease inhibitors and DTT to the cell lysate. I suspect that some of the glutathione beads are actually getting removed in the flow through fraction. Is it better to use Glutathione Sepharose beads instead of the GST- spin purification column?

What does it meaning By "incubated cell lysis in the spin column in an end-to-end rotator"? Does the lysate mix with the resin in the column? Usually, a spin column is prepacked.
For higher yield and more protein, check out the new bioreactors at https://qizhonglabs.com/main/.

#6 biolcrazy

biolcrazy

    member

  • Active Members
  • Pip
  • 16 posts
0
Neutral

Posted 17 July 2011 - 05:51 PM

sepharose is the brand name of the agarose from ge healthcare (nee pharmacia).


Thanks! i jus figured that out! :)

#7 biolcrazy

biolcrazy

    member

  • Active Members
  • Pip
  • 16 posts
0
Neutral

Posted 17 July 2011 - 06:18 PM


I am using the Pierce GST spin purification column for purifying my GST tagged protein. I have tried using the B-PER reagent/ sonication for bacterial cell lysis. I find that my protein goes in through the flow through fraction and I can't get any protein in the elution fraction. I incubated the cell lysate in the spin column at 4C in an end-to-end rotator for half an hour before spinning it around 1000 rpm to remove the unbound fraction. I add protease inhibitors and DTT to the cell lysate. I suspect that some of the glutathione beads are actually getting removed in the flow through fraction. Is it better to use Glutathione Sepharose beads instead of the GST- spin purification column?

What does it meaning By "incubated cell lysis in the spin column in an end-to-end rotator"? Does the lysate mix with the resin in the column? Usually, a spin column is prepacked.



Yes the column is prepacked. The protocol says that the lysate should be added to the column and incubated in an end-to-end rotator. However the resin gets dislodged in the subsequent centrifugation steps and it becomes difficult as the resin gets stuck in the middle of the column. When I just add more wash buffer and centrifuge to make the resin settle down I think some of glutathione beads get removed in the flow through and hence I lose protein in the flow through. I have tried avoiding incubating in the rotator and just let the lysate pass through (gravity flow) but lysate does not pass through and gets stuck. As soon as I centrifuge the column the resin gets dislodged.




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

©1999-2013 Protocol Online, All rights reserved.