Hi everybody,
I am dealing with protein naturally suspended in seawater. I filter freshly-sampled seawater volumes (about 10 L usually) through GF/F filters and extract the trapped proteinaceous material by filter homogenization and incubation in a common prot. solubilization buffer (Urea 7M, thiourea 2M, CHAPS 2%, DTT 1% and NDSB-201 + antiprotease cocktail 1%). I've had good results so far: I ended up with a fairly consistent amount of proteins, after TCA/acetone precipitation of the filter extracts, that eventually I've been able to run with 1D-GE and stain with coomassie.
However I'd like to extract proteins avoiding denaturation as much as possible, as I need it for further applications. I'm a newbie when it comes to this proteomic field but I know that many proteins are already in their non-native state or underwent degradation while suspsended in seawater, yet I'd like to limit further denaturation as I collect them. I've been suggested to use Native PAGE buffers for extraction and concentration but I'm not sure if it's gonna work in this case. Has naybody dealt with that previously? All ideas and suggestions are welcomed!
Thanks in advance!
Dear Paolo:
have you ever used tangencial filtration systems? This allows you to filter large volumens and finally you get the proteins in their "native" state.
Best wishes,
Rosa María
Dear Paolo:
have you ever used tangencial filtration systems? This allows you to filter large volumens and finally you get the proteins in their "native" state.
Best wishes,
Rosa María
Dear Rosa Maria,
Thanks for your reply. Actually I've never used tangential filtration, and after talking with my colleagues it does not seem to be the best solution for us.
Any other suggestion would be very appreciated.
Thanks again
I agree that tangential flow filtration is probably the best way to process such a large volume (10 liters). Ultrafiltration using pressurized cells, which uses much the same principle, could be used, but the usual laboratory cells are too small to be practical.
If the sample weren't so salty, you could capture the proteins on an ion exchange resin added to the container of water, pour the resin into a column, and elute it with a salt jump. Unfortunately, at the high ionic strength of seawater, many proteins won't bind.
But perhaps you can use the saltiness to your advantage by capturing the proteins on a hydrophobic interaction resin, such as Phenyl-Sepharose by the same bulk-binding method. In this case, you would actually increase the salt concentration by adding ammonium sulfate to 1 M, add the resin, mix it gently but thoroughly to allow the proteins to bind, pour the resin into a column, and elute the proteins with a salt-free buffer.
This would result in a concentrated protein fraction containing all the proteins that bound to the resin by hydrophobic interactions. You could also fractionate the proteins by eluting the column with a gradient of decreasing salt instead of in a single step.
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