I have crude protein extract that I want to purify on a SP-Sepharose column. Is it okay to filter the crude sample to get rid of suspended debris by vacuum. Can the foam generated by doing so, in fact be a sign of protein denaturation?
Hello Shivansh, I would suggest you run a cation/anion exchange column first instead of vacuum filtration because the filters are not cheap and you will need quite a number, even after all that, there will still be contaminants. The ion exchange columns do not need an affinity tag and can be used to remove a large proportion of proteins with isoelectric points higher or lower than that of your protein of interest. After running this column, the sample should be more suitable and ready for gel filtration; the gold standard step for protein purification.
Thanks for the suggestion. However, by vacuum filtration I mean removing the cell debris in my crude sample before I load it onto the Ion-exchange Column. Is it safe to do that or I should rather centrifuge the crude sample?
You can use a syringe filter to remove suspended particulates from your sample, but if you do you should use one with a glass fiber prefilter to help prevent clogging. This will avoid the foaming issue. With any filter, there will be some loss of protein due to it sticking to the filter, so you should use the smallest filter that is able to process the whole sample before clogging.
Instead of filtration, you could use centrifugation to clarify the sample. This avoids the clogging and sample loss issues.
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