Hello! I am reviewing a procedure for exosome/EV purification from a large-volume blood sample (porcine, terminal draw). In brief, protocol is:

1) Blood collection

2) Two-step spin to get platelet-poor plasma

3) PEG precipitation (ExtraPEG procedure)

4) Pellet --> Sephadex G-100 --> concentrated via dialysis

To elaborate, here's their quote for Step 4:

"To eliminate protein aggregates and fibrin filaments, the redissolved PEG precipitate was passed through a Sephadex G100 column. The resulting fractions were pooled, concentrated via overnight dialysis, and stored appropriately depending on the intended use."

That explanation for Step 4 has me confused. Could use some help interpreting this. Here are direct questions:

Q1) Sephadex G-100 can separate in the 5-600 kDa range (according to Cytiva Life Sciences), so exosomes/EVs should show up in the void volume, correct?

Q2) Is there an unstated purposed to remove PEG 6K from the precipitation?

Q3) Would a Zeba column (40 kDa MWCO) serve the same purpose?

Thanks for your help!!!

PS - For reference, the protocols using size exclusion chromatography for EV separation use Sepharose CL-2B (2% agarose; sep range for 70-4000 kDa)... so quite a bit different from the Sephadex G-100

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