I am currently researching a membrane-based method for virus purification, and I would like to compare it in economic terms to Density gradient centrifugation (DGC) in industrial scale. Which capacity can DGC handle in terms of viral sample volume and concentration? What are the typical process times? And which viral concentration can the method achieve at the end?
For ultracentrifugation, some machines such as Beckman ultracentrifuges can do continuous flowthrough into & out of a special rotor for high-throughput academic or private industry uses. Volume/capacity varies if using a flow-through system versus tubes; tube sizes depend on rotor, which affects max rotational speed.
We typically use an Opti-prep density gradient with a Beckman SW-41Ti rotor at 40,000 RPM for 2h (with no brakes, putting it at 2.75-3h) at 4ºC to process up to 6 tubes (paired matching) with 1.5 mL volume of virus (we start with Amicon-concentrated virus) per tube (9 mL total volume possible).
We normally see a virus band when we have sufficient protein to start the purification. If you are unsure of which layer your protein/particle of interest is, do a section-by-section QC such as WB + silver staining and/or [capture] ELISA (we divide each tube up into 13x 1 mL layers taken top-to-bottom, then WB + silver stain each layer).
You can concentrate afterwards as well, but for practical reasons like volume reduction, we concentrate beforehand. You can also do a sucrose concentration for bigger batches.
For ultracentrifugation, some machines such as Beckman ultracentrifuges can do continuous flowthrough into & out of a special rotor for high-throughput academic or private industry uses. Volume/capacity varies if using a flow-through system versus tubes; tube sizes depend on rotor, which affects max rotational speed.
We typically use an Opti-prep density gradient with a Beckman SW-41Ti rotor at 40,000 RPM for 2h (with no brakes, putting it at 2.75-3h) at 4ºC to process up to 6 tubes (paired matching) with 1.5 mL volume of virus (we start with Amicon-concentrated virus) per tube (9 mL total volume possible).
We normally see a virus band when we have sufficient protein to start the purification. If you are unsure of which layer your protein/particle of interest is, do a section-by-section QC such as WB + silver staining and/or [capture] ELISA (we divide each tube up into 13x 1 mL layers taken top-to-bottom, then WB + silver stain each layer).
You can concentrate afterwards as well, but for practical reasons like volume reduction, we concentrate beforehand. You can also do a sucrose concentration for bigger batches.
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