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Question about MBP purification

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#1 skepticalguy99



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Posted 12 December 2014 - 08:20 AM



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!




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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



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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.

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