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selection of the media for ion exchange chromatography

ion exchange column

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#1 chn09

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Posted 31 December 2013 - 09:37 AM

Dear friends,

 

I am trying to purify a protease enzyme from Bacillus sp. The predicted pI of the protease is 9.2 and i am using the culture supernatant as the source of the enzyme. I have used ammonium sulfate precipitation and checked the activity of the enzyme

 

My queries are

 

1. What ion exchange column can i choose for the ion exchange chromatography? Cation or anion exchange?

 

2. How can the column packing be done manually? I do not have prepacked columns or FPLC apparatus. Can the packed column be operated using a syringe or peristalitic pump?

 

 

 

 



#2 mdfenko

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Posted 31 December 2013 - 03:11 PM

1: if you want to use buffers around neutral pH then use a cation exchanger (recommended). if you want to use an anion exchanger then you'll have to use a pH higher than the pI (not recommended).

 

2: ge healthcare has handbooks available (free for download) which describe how to pack and run classical lc. i recommend "gel filtration" (for how to pack and run classical lc)  and "ion exchange chromatography" for how to run ion exchange (medium selection, etc). appendix 3 in "ion exchange" and appendix 1 in "gel filtration" tell you how to pack a column.

 

there are also other useful handbooks available.


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