Cesium Chloride Density Gradient Ultracentrifugation Workflow

We understand the importance of your upstream conditions and the critical nature of optimizing both the cell media and transfection reagent utilized. Our team of scientists, process engineers, manufacturing specialists, and quality control experts are here to help define and test custom reagents for your AAV needs.

Get in touch with our team to discuss the best solution for your specific application.

Following lysis, endonuclease treatment, and the clarification of the culture harvest, precipitation can be used to reduce impurities, including proteins and nucleic acids, and to concentrate the sample prior to ultracentrifugation.

PEG-8000 with Sodium Chloride (NaCl) is ideal if you are using a single precipitation step method. For a two precipitation step method, you can use Calcium Chloride as a precipitant, followed by PEG-8000 with NaCl. We have off-the-shelf stocks of 1 M Calcium, 40% PEG-8000, and 2.5 M NaCl in stock and ready to ship.

Cesium Chloride (CsCl) forms a linear density gradient under ultracentrifugation that is used to separate empty from full AAV capsids into distinct bands.

There are multiple approaches to prepare the CsCl gradient for AAV purification:

• Directly mixing the AAV sample with a saturated solution (1.9 g/mL).
• Overlaying the AAV sample over a single CsCl solution (1.4 g/mL).
• Using a double layer approach with two different CsCl concentrations (1.5 and 1.3 g/mL).

Using dialysis, the CsCl-containing buffer will exchange with the dialysate to a more suitable solution for either storage or continued preparation. We have multiple options to perform dialysis using your buffer solution of choice, including 1X PBS with 5% Sorbitol or 50 mM HEPES (pH 7.4), 0.15 M NaCl, and 25 mM EDTA (HNE) buffer.

We have extensive experience manufacturing custom storage buffers. Our team of scientists, process engineers, manufacturing specialists, and quality control experts are here to help define custom storage reagents to meet your needs.

Fill out our custom order form or get in touch with our team to discuss the best solution for your specific application.