I have EPS from lactic acid bacteria and I want to remove carbohydrate residues from medium. I should say I will do these steps befor using size exclusion chromatography:
centrifugation
TCA perticipation
centrifugation
ethanol percipitation
dialysis
lyophilization
size exclusion chromatography
could u tell me that doing size exclusion is necessary or not?
Small carbohydrate (and other) molecules can be removed during the dialysis step, with the size that can be removed depending on the molecular weight cutoff of the dialysis membrane. For example, a 10,000 MWCO membrane might effectively remove molecules of a few thousand molecular weight, given sufficient time. Size exclusion chromatography can be used to fractionate larger polysaccharides based on size (and shape), and will also effectively remove any remaining small molecules. It's up to you to decide how much purification is needed for your experiments.
What if, if I dont conduct dialysis step for my EPS? I do not have dialysis bag for now.
I wanna conduct NMR analysis, do you think there will be any drawbacks?
Thanks
Best
For NMR analysis, a highly pure sample is pretty important. Otherwise the spectrum will be impossible to interpret. If dialysis is needed to assure sufficient purity, then you should either do it or use another purification method that can replace it.
Let's consider step by step;
-Centrifugation; is necessary for removing the non-soluble pellet,
-TCA participation; for further investigation, if you are targeting to use SEC you should deplete your proteins otherwise they will interfere with your target in SEC due to similar MWs.
-centrifugation ethanol precipitation; After TCA precipitate, you can remove the protein and sequential ethanol application can concentrate the polysaccharides and nucleic acids. That seems to be ok for the enrichment and clean up
-dialysis; Up to this step, you may probably remove the major part of the carbohydrate residues. Dialysis is not necessary in my opinion and if you perform SEC (gel filtration) you can also get rid of all small molecules due to the separation mechanism and you don't need to apply additional step such as dialysis if you won't use SEC resins for getting higher recoveries and time-efficient method, I suggest to use ultrafiltration with proper MWCO.
lyophilization; Necessity of this step depends on your analyte recovery and requesting concentration but also for stability concerns.
size exclusion chromatography; If you perform these steps I guess additional SEC is not required but you may perform the only one injection to validate pretreatment of this approach.
- Badges
- Science topic
- Similar topics
- Biological Science
- Biochemistry
- Enzymology
- Enzymes