#1 How Do You Best Prepare Your Protein Samples for Purification?
A popular way to clarify lysates prior to capture step is to centrifuge at 45,000 rpm, 30 min, with a 45 Ti rotor or equivalent.
When filtering proteins, be sure to select a low protein binding membrane such as PVDF or PES. Nylon fliters often bind protein and will cause a loss of yield.
Samples should be 0.45 or 0.2 µm filtered to remove particulates and small contaminants. If your sample does not flow easily through one of these filters, try a 0.8 µm filter. This size often works well for proteins with visible particulates and proteins not amenable to smaller pore filtration.
When preparing your sample for ion exchange, dilute or desalt it to bring its conductivity below 10 mS/cm. This will ensure optimal binding.
Use proper protein storage methods after purification to protect proteins.
- Short term (24 hr or less): 4°C
- Long term: –80°C. Include 5–50% glycerol, albumin (10 mg/ml), and/or reducing agents like DTT or BME in the storage buffer. Freeze protein in small aliquots in microcentrifuge tubes. Flash freeze tubes using liquid nitrogen, or dry ice/ethanol bath
- Can also store protein as ammonium sulfate precipitate at 4°C
This tip is from the Chromatography Success Guide. The Chromatography Success Guide provides tactical advice on preparative chromatography and protein purification.