I was wondering if one can use an Amicon column to get rid of proteins above the MWCO, by collecting the filtrate instead of the retentate. My protein of interest from affinity chromatography purification is 13kDa but an SDS-PAGE revealed faint bainds above 30 kDa, which I would like to get rid of.
Has anyone ever done this ?
Yes this is possible, and yes I've done this.
It feels a little bit like 'cheating', but I can't argue with it. The only thing I'd say is that there's a risk of losing some protein as it might get trapped in the filter membrane. It was never an issue for me as we had high-yield, but if your yield is low this may be more problematic.
Best wishes,
James
You can, and yes we have done this. However, your impurity and target protein have very close MWs. A typical rule for using ultrafiltration membranes is to use MWCO 1/3 of your retentate. The only membrane you can use is 30kd, and depending on the actual MW of your impurity, you may end up having some of it in your filtrate.
The issue may be complicated by the presence of aggregates/oligomers of either the target protein or the impurities. As usual with proteins, nothing beats trying what applies to your particular case: run a trial with a 30 kDa MWCO cartridge and look at the outcome by SDS PAGE
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