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Tech Note

Mega-scale adenovirus purification system

Our Adeno-X Mega Purification Kit allows you to purify large amounts of recombinant adenovirus in 1.5 hours or less. With a simple protocol and a powerful syringe pump, you can purify up to 1013 viral particles without affecting infectivity. Adeno-X purification achieves nearly complete recovery of the infectious particles present in your crude supernatant, and the purified stocks are stable for years at –70°C. The kit is excellent for any application requiring mega-scale amounts of purified adenovirus, starting from cultures of twenty-five 15-cm dishes.

  • Easy-to-use, streamlined protocol
  • Chromatographic method eliminates messy CsCl gradients
  • No more handling of large culture volumes
Introduction Protocol overview Results Conclusions

Introduction  

Large-scale simplicity

The kit includes two filter assemblies and all the buffers needed to make two mega preps. All you need is a reliable syringe pump to provide automatic and consistent flow rate control so that your prep works every time (we recommend the GENIE Plus syringe pump from Kent Scientific). The straightforward protocol allows you to purify the adenovirus directly from the cell pellet, eliminating time-consuming filtration of the culture supernatant, which contains only small amounts of virus (~2% of that found in the pellet). You simply pellet the cells when the cytopathic effect (CPE) is nearly complete, lyse the cells, and purify the virus.

Protocol overview  

Virus chromatography is quicker & easier than CsCl gradients

As with all of our Adeno-X purification kits, the virus is purified chromatographically, using a unique membrane that selectively binds adenoviral particles. Unlike complicated CsCl gradients, chromatographic purification is safe, nontoxic, and requires no advanced training or expensive ultracentrifugation equipment. The virus-binding membranes are housed in a single-use, multi-membrane cartridge assembly that fits securely on a standard size BD Luer-Lok syringe. For added convenience, the purification assembly comes preassembled, sterile, and ready to use.

A convenient one-way valve makes purification easier by eliminating the need to disconnect the syringe for refilling during the binding and washing steps. Liquids are brought into the syringe through the one-way valve merely by withdrawing the plunger. Depressing the plunger drives the solutions through the purification cartridge—it couldn't be easier!

All you need is a powerful pump to make the procedure rapid and easy

From beginning to end, the entire procedure is very fast and straightforward (Figure 1). Once the CPE is nearly complete in your amplification culture, you harvest and lyse the cells (to release the virus) and prepare the lysate for purification. To collect the virus, you can use a syringe pump to drive the clarified lysate through the three-tiered purification cartridge. The pump provides consistent pressure and flow rates to foster more efficient virus binding and excellent virus recovery, and eliminates the need to control the flow by hand. Bound virus particles are washed once and then eluted from the membrane using a convenient volume of elution buffer. The streamlined protocol, from harvest to elution, takes just 1–1.5 hours to complete.

Adeno-X Mega purification kit protocol

Figure 1. Adeno-X Mega Purification Kit protocol. From a large-scale culture of infected HEK 293 cells (~25, 15-cm plates), cells are collected and lysed by multiple freeze-thaw cycles. Nucleic acids are digested to reduce viscosity, then the lysate is clarified and passed through the purification filter assembly. After a brief wash, purified virus is eluted and ready to be used, titered, or stored. The entire process takes less than 1.5 hr.

Results  

Achieve high titers of high quality virus

The titer and purity of virus produced by our Adeno-X purification kits are comparable to those achieved by CsCl density gradient centrifugation. The purified virus yields are characterized by excellent recoveries and high infectivity (Figure 2). In adenovirus preparations, not all particles are completely formed and infectious. With Adeno-X purification kits, ratios of virus particles to infectious units (IFU) can be as low as 20:1. Depending on the adenoviral backbone used and the nature of the expression cassette, the Adeno-X mega kit can yield up to 1013 adenoviral particles, which potentially corresponds to as much as 5 x 1011 IFU. A DsRed-Express ViraTrak virus purified with Adeno-X technology efficiently infects HEK 293 cells, which undergo a timely CPE and produce high levels of DsRed-Express protein (Figure 3).

Adeno-X Mega Purification yields nearly quantitative recoveries

Figure 2. Adeno-X Mega purification yields nearly quantitative recoveries. HEK 293 cells were seeded and infected with Adeno-X ViraTrak DsRed-Express virus, and harvested when the cytopathic effect was nearly complete. The resulting adenovirus was then purified according to the Adeno-X Mega Purification Kit protocol. Total infectious units (IFU) ± standard deviation for each fraction (crude, flowthrough, wash, and eluate), were determined by infecting fresh HEK 293 cells with dilutions of each fraction, and counting the fluorescent cells under a fluorescence microscope 48 hr later. Virtually all of the starting material was recovered in the eluate as purified virus. Purified virus demonstrated greater apparent infectivity than the virus present in the crude lysate. The process was efficient and fast, allowing 1.23 x 1011 adenoviral IFU to be purified in 1.5 hour.


Progression of the CPE and fluorescent protein expression in infected HEK 293 cells

Figure 3. Progression of the CPE and fluorescent protein expression in infected HEK 293 cells. HEK 293 cells seeded on a 6-well plate were transduced with an Adeno-X ViraTrak DsRed-Express adenovirus at an MOI of ~1–2. During the following 4 days, the CPE (top row) and DsRedExpress expression (bottom row) were monitored by phase contrast and fluorescence microscopy, respectively. By day 4, the CPE was nearly complete and correlated with high levels of DsRed-Express protein.

Conclusions  

All of our Adeno-X purification kits are characterized by outstanding virus recovery, and produce highly purified and highly infectious adenovirus with low viral particle/IFU ratios. The Adeno-X Mega Purification Kit simplifies adenoviral purification and provides large-scale convenience at a great price.

Related products

Cat. # Product Size License Quantity Details
631032 Adeno-X™ Mega Purification Kit 2 Preps USD $1108.00

The Adeno-X Mega Purification Kit is achromatography-based purification system for concentrating and purifying infectious adenovirus. The kit provides all ofthe buffers and filters needed to purify adenovirus from cell lysate. This Kit should be used with a continuous flow syringe pump (see User Manual for details).

Notice to purchaser

Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval.

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Back

The Adeno-X Mega Purification Kit protocol

The Adeno-X Mega Purification Kit protocol

The Adeno-X Mega Purification Kit protocol. From a large-scale culture of infected HEK 293 cells (~25, 15-cm plates), cells are collected and lysed in multiple freeze-thaw cycles. Nucleic acids are degraded to reduce viscosity, and the lysate is clarified and passed through the purification filter assembly. After a brief wash, purified virus is eluted and ready to be used, titered, or stored. The entire process takes less than 1.5 hr.

Back

The filtration apparatus of the Adeno-X adenovirus purification kits are preassembled and includes a one-way valve to simplify the purification process

 The filtration apparatus of the Adeno-X adenovirus purification kits are preassembled and includes a one-way valve to simplify the purification process

The filtration apparatus of the Adeno-X adenovirus purification kits are preassembled and includes a one-way valve to simplify the purification process. The one-way valve eliminates the need for repeated syringe removals when binding and washing. The adenoviral stock is brought into the syringe through the valve and pushed out through the purification filter cartridge by the syringe plunger.

Back

Adenovirus is purified directly from the cell pellet of HEK 293 cells

Adenovirus is purified directly from the cell pellet of HEK 293 cells
Adenovirus is purified directly from the cell pellet of HEK 293 cells. Starting from the second day after infection, the vast majority of virus is found inside the cell, not in the supernatant. Therefore, combining the supernatant with the cell pellet (as with competitor protocols) unnecessarily complicates the protocol without increasing the amount of virus purified.

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631032: Adeno-X Mega Purification Kit

631032: Adeno-X Mega Purification Kit


Adenoviral product selection guide

Adenoviral selection guide

Browse our adenoviral selection guide to choose the best system for your needs. We offer both constitutive and Tet-inducible expression systems based on the Adeno-X adenoviral system 3, an advanced gene delivery system that quickly and efficiently produces adenoviral particles.

Selection guide Product finder tool

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