Dear experts,
I am planning a clean-up step in a protein purification process where I actually thought of using Sephacryl S200. In the stock room, I found some Sephadex G200 and thought, I'd give it a try, despite pretty old and not commercially available anymore.
GE states in the S200 manual, that the Sephadex flavour would require much longer equilibration times (advertising, that S200 would be much speeding up the process).
Anyone out there has some real life experience and could comment this issue? And despite ~ 4 times the price of the loose material (regarding S200, if I should have to resort on that), would it make sense to buy a pre-packed 60cm column instead of packing my own?
Thanks for you help!
depends a lot on your separation problem: Do you want to separate proteins that are very different in size or are they similr ?
Efficiency of gel filtration method depends a lot on the quality of the column package, therefore if you dont have experience and tools for packing a column yourself it might be a good idea to buy a pre-packed column. Keep in mind that you also would need the right system/pumps to run the column.
Hi
besides your old material, which might still perform well you will need proper equipment to pack a 60cm column. That is usually done by attaching a second column, fill both with the loose material and then pack under pressure. It is not that straightforward to reach a good packing for decent protein separation. If you have some funding left by the end of the year a prepacked column might be a good way to invest it.
Cheers
Hi Wolfgang,
the main difference is in the carrier material. The Dextran based can be used with low pressures only (10 cm H2O should be not exceeded). So also, the flow rates are much lower. On the other site, the separation effect is somewhat better (depending on the differences in molecular weights). Self packing is a little bit more difficult due to the pressure limits.
The acryl based carrier allows higher pressures, higher flow rates and the handling is much easier.
The main questions are molecular weight differences and column load and this results in the answers to the columns diameter and length. Both materials are easy to clean in place, they can be used several hundred times.
Just one last remark: don’t forget the outgassing for both, the gel and the buffer.
Thanks to everyone for your suggestions! Just orderd the Sephacryl column.
The cleanup problem is separating a ~60kD band (a putative dimer) from the desired 30kD monomer, with some more (probably proteolytic) bands present (they all elute at the same conditions in IMAC).
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