Dear Jenny

In principle you can certanilly perfrom cation or anion exchange by gravity flow. The main issue is that with gravity yuo cannot perform a linear gradient in salt concentration but you have to prepare several buffers with increasing concentrarion of salt and perform a multistep gradient.

I have done this in small scale several tine in the past using CM (for cationic exchange) or DEAE (for anioic exchange) FF resin of GE to perform a pre-screeining to compare the expression and solubility level of several E.coli strain expressig the same protein in different culture condition.

Sinbcerelly I never used the Nuvia S resin. The resin appliability to gravity flow depends principally from the resisntanc of the resin to the flow. Fast flow resin are preferable because otherwise the flow will be very slow also with small amount of resin. However the best way, is just to try and see how fast is the flow with water once you assembled the coloumn. If the flow is very slow also with water, you have a problem.

p.s: filter at 0.22um the sample before to be loaded in the coloumn, because if your sample is not well centrifuged, yoru coloumn flow may stop and you will be not able to go on with the purification.

Regarding empty coloumn to be used, generally for small resin volume up to 200ul i like the Biorad Bio-Spin coloumn cod.7326008) from 200ul to 500ul the Biorad Poliprep (cod. 7311550) and for resin volume higher than 1ml the PD-10 empty coloumn (GE cod. 17043501)

You have to choose the format that is the best for you on the basis of the amount of protein that you expect to produce from your culture. eg. 100ul resin for 0,1-0,2 mg. 2ml resin >2-5mg alspo in relation of the binding capability of your specific resin.

good luck

Manuele

Thank you, Manuele Martinelli !

I agree with everything Manuele wrote.

However, one can do linear gradients, no problem, done it in the distant past before the automated systems became available.

Two beakers connected via a liquid bridge, e.g., a piece of tubing filled with Buffer A. The beaker with A is stirred and the liquid is taken from this container to the column. As it empties, it draws B from the second vessel.

If you are working in an old established lab with lots of old equipment still hiding in closets, you may be able to find somewhere a gradient mixer. The same thing as the beakers, but fixed together and with a fixed exit near the bottom of the vessel with buffer A.

Just checked, Sigma-Aldrich is still selling gradient mixers.

Yes. You can do first SBC (static binding capacity) experiments followed by different concentration of salt wash or different pH wash or both together as per your requirement and desired product stability. You can do it manually. However, Chromatography system's run will be very robust , reproducible, data recording and interpretation very much easy.

Hope this helps.

Best Regards,

Yamak

http://www.bio-rad.com/en-in/category/empty-gravity-flow-columns?ID=66878000-baac-4a58-8a3c-9887c3825dd1