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
- - - - -

How to make antibody based affinity column for protein purification

antiboy affinity protein purification cojugation Scale up

  • Please log in to reply
4 replies to this topic

#1 ionchannelbk

ionchannelbk

    member

  • Active Members
  • Pip
  • 14 posts
1
Neutral

Posted 21 September 2017 - 05:52 AM

Hello,

 

I am trying to purify a protein from heterologous expression in yeast system using this protein specific antibody. I tried antibody crosslink to Protein A/G beads by DSS, but did not work. Anyone knows how to make your own antibody based affinity column? What is the most efficient products to make this column? Can I easily scale-up the purification?

 

Thanks in advance!



#2 labtastic

labtastic

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 201 posts
20
Excellent

Posted 22 September 2017 - 09:18 AM

Simplest way is probably to buy some NHS-activated sepharose, and couple your purified antibody to it. Scaling up is simple as long as you can afford the NHS-sepharose (~10$/ml from GE Healthcare).

 

Another option is CNBr activated sepharose (also not super cheap_.

 

Cheapest option is to buy standard Sepharose 4B resin and activate it with CNBr yourself prior to coupling. Protocols for this can be found easily online. Practice first on some cheap protein (BSA, GFP etc) to get the process down. And work in a fume hood.


Edited by labtastic, 22 September 2017 - 09:54 AM.


#3 mdfenko

mdfenko

    an elder

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 3,246 posts
198
Excellent

Posted 22 September 2017 - 02:24 PM

the problem with coupling with nhs or cnbr sepharose is that it doesn't force proper orientation of the antibody and your affinity gel will not be as efficient as possible. i've had great success with the pierce protein a antibody orientation kit (a bit pricey but worth it for me). if protein a doesn't work well then you can try the protein g antibody orientation kit. check the manuals (links on the pages for the products) to determine which might be better for you. they use dss but the system is well defined.


Edited by mdfenko, 22 September 2017 - 02:26 PM.

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

#4 ionchannelbk

ionchannelbk

    member

  • Active Members
  • Pip
  • 14 posts
1
Neutral

Posted 27 September 2017 - 06:14 AM

Thanks Veteran and mdfenko for information. I read NHS-activated sepharose manual. It cross-links small amino containing protein. Does it work for big protein like antibody? Thanks.



#5 mdfenko

mdfenko

    an elder

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 3,246 posts
198
Excellent

Posted 27 September 2017 - 02:22 PM

it should but, like i said, it won't necessarily orient all of the antibodies so that they're available to bind antigen.


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.