Hy everyone,
I'm facing some troubles when changing the buffer of my protein solution. I'm doing a 2-step purification of my protein, first on a nickel-column and second on a heparin column. Between the two steps I add glycerol to the protein solution in order to freeze it until further use. I need to remove the glycerol and the salt from the former buffer before adding the protein to the heparin column and to achieve this I'm using the Amicon® Ultra-15 Centrifugal Filter Units. However, it looks like this is not the best way to do it, since this process always takes ages and my protein starts to aggregate at the membrane resulting in little flakes in the solution. I guess the aggregation makes it even harder to perform the filtration since the aggregates block the pores of the membrane.
I was thinking about using those Amicon® Stirred Cell models, but I have no experience with them and they are pretty pricy. Can some of you share your experience about the stirred cells with me?
I would be also very happy to hear about other alternatives for buffer exchange that work great!
Thank you very much in advance.
All the best,
Pascal
PS: In case you need to know the compostion of the buffer:
- 50 mM Na-PO4
- 0,5 M NaCl
- 84,6 - 400 mM Imidazol
- 10% Glycerol
Hi there,
I would avoid the freezing step to skip the glycerol addition and use dialysis instead of filtration so that the concentration of protein remains constant during the buffer exchange. The issue might be that your protein is actually sensitive to the decrease of salt concentration and/or sensitive to the increase iin protein concentration. Does aggregation happen early in your process (ie. before the decrease in salt concentration)?
Thank you both for your answers!
Dimitrios Latousakis
: Yes, the PD10 columns might be a good idea. Do they work with glycreol well?About the dialysis membranes: Last time i used them another Protein I analysed was also aggregating inside those membranes. So I'm not super convinced about these. However they might still be an option for this protein.
Dominique Liger : I actually tried to avoid the freezing step last time and didn't add glycerol before buffer exchange. However, even then the solution was really a pain to exchange. Probably because the protein formed a layer over the membrane and made it really hard to filter the solution. By pipetting I could actually remove visible flakes of protein that sticked to the membrane.
Aggregation happens only during buffer exchange, but very early. Basically before adding the new buffer. So yes, might be a problem with the combination of decrease in salt and increase of protein.
I really want to avoid the dialysis because it takes a day (correct me if I'm wrong). But if there is no other good option I will try it.
Thank you very much Dimitrios Latousakis
. I will definitely consider your suggestions.
Hi again,
Dialysis may be much shorter (a couple of hours) if the exchange buffer is stirred, if you renew several times the exchange buffer (every 2 hours) and if volume ratio between dialysate and exchange buffer is at least 1:200.
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