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
- - - - -

Question about MBP purification


  • Please log in to reply
2 replies to this topic

#1 skepticalguy99

skepticalguy99

    member

  • Members
  • Pip
  • 1 posts
0
Neutral

Posted 12 December 2014 - 08:20 AM

Hi,

 

I'm currently working with a MBP-fusion to my protein of interest.  I'm mostly curious about the resin that is utilized for purification.  After expression in E.coli, purification on amylose resin is very common.  However, the resin begins to degrade after a few uses and I'm wondering if anyone knows any more sustainable alternatives?  I've heard about using a His-Tag as the main purification method, but I had also looked at Dextrin Sepharose resins.  The problem is I haven't had any luck with a literature search on Dextrin stability in the presence of the amylases in E.coli extracts.  Any help or suggestions would be greatly appreciated!



#2 CPRES

CPRES

    Cellcounter

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 140 posts
19
Good

Posted 28 January 2015 - 04:29 AM

Here is a big list of available protein binding resins.


So. Now that you have your first ever question on bioforum answered (or not), mail yourself your username and password so you don't forget them, and then come back soon to update us on how it all worked out. That's how you build Karma in science.


#3 labtastic

labtastic

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 88 posts
4
Neutral

Posted 28 January 2015 - 08:24 AM

His-tag is the most commonly used tag for protein production facilities largely because (i) His tags are small and easy to encode in a protein, and rarely interfere with protein function, (ii) the resin has high binding capacity (>10 mg protein/ml resin in most cases), (iii) the reagent needed to elute proteins is cheap (imidazole) and (iv) the resin can be regenerated and re-used for years and years, you just need to clean after each use (EDTA, Guanidine, NaOH, Ethanol...rebind the Ni...store in 20% ethanol).

 

The only down side to His-tag columns is that your protein doesn't always come out as pure as a lot of other affinity resins, particularly with poor expressing proteins. Just means you need to do additional purification steps (ion exchange, gel filtration) if you need it cleaner.

 

Or go old-school...don't use any tags and purify native. smile.png


Edited by labtastic, 28 January 2015 - 08:25 AM.





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

©1999-2013 Protocol Online, All rights reserved.