selection of the media for ion exchange chromatography - (Dec/31/2013 )
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?
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.