Transduction assay of AAV3 standard material (eGFP) using HeLa cells. HeLa cells (50 µl) were plated in 96-well plates at 200,000 cells/ml in DMEM + 10% FCS. 50 µl of eGFP-filled AAV3 capsids (3.1E+10 vg/ml) were added to the cells and incubated for 48 h at 37°C and 5% CO2. After incubation GFP-expression was visualized with a fluorescence microscope.
SDS PAGE with AAV3 eGFP-filled capsids. The AAV3 VP1, VP2 and VP3 proteins were separated on a 10% SDS PAGE and visualized by Pierce Silver stain kit. Only VP1, VP2 and VP3 proteins in the correct stoichiometry of 1:1:10 are detectable indicating a purity of the AAV preparation of >95%.
Transduction assay of AAV3 standard material (eGFP) using HeLa cells. HeLa cells (50 µl) were plated in 96-well plates at 200,000 cells/ml in DMEM + 10% FCS. 50 µl of eGFP-filled AAV3 capsids (3.1E+10 vg/ml) were added to the cells and incubated for 48 h at 37°C and 5% CO2. After incubation GFP-expression was visualized with a fluorescence microscope.
SDS PAGE with AAV3 eGFP-filled capsids. The AAV3 VP1, VP2 and VP3 proteins were separated on a 10% SDS PAGE and visualized by Pierce Silver stain kit. Only VP1, VP2 and VP3 proteins in the correct stoichiometry of 1:1:10 are detectable indicating a purity of the AAV preparation of >95%.
- Fully assembled AAV3 capsids filled with an eGFP reporter gene controlled by a CMV promoter
- Serotype-specific conformational epitopes
- Reliable positive control for dot blot, western blot, ELISA, PCR and cell-based assays
- Quality controlled for purity, filling grade, aggregation, total capsid and genome titer
- Alignment to internal reference standard material
For US shipment, the packages are only sent out for delivery on Mondays and Tuesdays, in the EU from Monday until Wednesday.
Product description
| Filling grade | > 60% full capsids, please find the lot-specific filling grade on the CoA |
|---|---|
| Quantity | 100 µl |
| Application | Calibration of instruments e.g. mass photometry, Cell-based assay, Dot blot, ELISA, WB, ddPCR, qPCR |
| Purification | Affinity Chromatography (POROS CaptureSelect AAVX Affinity Resin, Thermo Fisher Scientific), Iodixanol gradient centrifugation for removal of empty capsids using OptiPrep (Cat. No. 1893, PROGEN) |
| Storage | Up to 2 weeks: 2-8°C; long term storage in aliquots at -80°C; avoid > 5 freeze/thaw cycles |
| Intended use | Research use only |
| Concentration | > 1.0E+12 vg/ml; please find the lot-specific concentration (total capsid and viral genome titer) on the CoA |
| Formulation | PBS + 0.014% Tween20 + 1 mM MgCl2 + 2.5 mM KCl |
| Source | Produced in HEK293T cells |
| Purity | > 95% by SDS-PAGE and silver stain |
| Quality check | Total capsid titer was assigned using AAV3 ELISA kits (PRAAV3R, calibrated with internal reference material) and viral genome titer by qPCR using eGFP primers; QC included analysis of filling grade, purity, aggregation, and endotoxin testing. |
| Transfer Vector | pAAV-CMV-eGFP-WPRE |
| Packaging Plasmid | pRep2-Cap3 + pHelper |
| Endotoxin | < 1.0 EU/ml (detection limit 1.0 EU/ml) |
| Note | Please centrifuge before opening to ensure complete recovery of vial contents; aliquoting and repeated freeze/thaw cycles can lead to a drop in titer |
Applications
| Tested applications | Tested dilutions |
|---|---|
| Dot Blot | Depending on primary antibody and detection method |
| ELISA | As a positive control in ELISA, dilute in ASSB 1x (provided with PROGEN’s AAV3 ELISA), analysis at least in duplicates is recommended |
| Cell-based Assay | Depending on the experimental setup |
| PCR | As standard or positive control in qPCR or ddPCR - concentration depending on experimental setup |
| Western Blot (WB) | Depending on primary antibody and detection method |
Background
Our AAV3 standard material consists of fully assembled AAV3 capsids with an eGFP reporter gene controlled by a CMV promotor. Final concentrations (total capsids and viral genome titer) are lot-specific and can be found on the CoA.
The AAV3 standard material is provided with a titer above 1.0E+12 viral genomes/ml in a liquid formulation. Since the buffer does not contain any stabilizing proteins or dyes, the capsids can be used in various applications, including dot blot, western blot, ELISA, qPCR, ddPCR and cell-based assays. Our material contains only a very low remaining amount of iodixanol allowing DLS-SLS-UV/Vis analysis. The lot-specific titers were assigned using AAV3 ELISA (PRAAV3R) according to our internally established standard material* and PCR using eGFP primers. Our comprehensive quality control ensures well-characterized capsid material, which can be implemented as reference material in a variety of assays to prove the validity of the corresponding assay.
PROGEN provides eGFP-filled AAV standard material for the AAV serotypes 1, 2, 3, 5, 6, 8, 9, rh10, and rh74.
*Our internal standard material for each serotype was characterized according to the protocol described in our poster Developing Reliable AAV Standards for ELISA (available in the downloads tab). Data on the establishment of standard material for specific serotypes can be found as part of the performance data for the corresponding ELISAs or can be provided upon request.
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Please have a look for the table below to see the combination of plasmids used for the production of our AAV standard material:
| Serotype | Transfer vector | Packaging plasmid | Helper plasmid |
| AAV1 | pAAV-CMV-eGFP-WPRE | pDP1rs | - |
| AAV2 | pAAV-CMV-eGFP-WPRE | pDP2rs | - |
| AAV3 | pAAV-CMV-eGFP-WPRE | pRep2-Cap3 | pHelper |
| AAV5 | pAAV-CMV-eGFP-WPRE | pDP5rs | - |
| AAV6 | pAAV-CMV-eGFP-WPRE | pRep2-Cap6 | pHelper |
| AAV8 | pAAV-CMV-eGFP-WPRE | pRep2-Cap8 | pHelper |
| AAV9 | pAAV-CMV-eGFP-WPRE | pRep2-Cap9 | pHelper |
| AAVrh10 | pAAV-CMV-eGFP-WPRE | pRep2-Caprh10 | pHelper |
| AAVrh74 | pAAV-CMV-eGFP-WPRE | pRep2-Caprh74 | pHelper |
High virus titers > 1.0E+12 vg/ml and liquid formulation (PBS with 0.014% Tween, 1 mM MgCl2 and 2.5 mM KCl) allow the use as a reference material in a variety of applications e.g. PCR, BLI, SEC-MALS, mass photometry, infectivity and many more.
The AAV capsids titer is measured by our PROGEN ELISAs and the genomic titer is measured by qPCR using eGFP primers.
Our AAV standard material contains the most commonly used and well known combination of an eGFP transgene with a CMV promoter
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