AAV9 standard material (eGFP)
Transduction assay of AAV9 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 AAV9 capsids (3.6E+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.
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SDS PAGE with AAV9 eGFP-filled capsids. The AAV9 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%.
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Transduction assay of AAV9 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 AAV9 capsids (3.6E+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 AAV9 eGFP-filled capsids. The AAV9 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 AAV9 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 | > 70% 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 AAV9 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 AAV9 ELISA kits (PRAAV9 and PRAAV9XP, 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-Cap9 + 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 AAV9 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 AAV9 standard material consists of fully assembled AAV9 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 AAV9 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 AAV9 ELISA (PRAAV9 and PRAAV9XP) 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
Related Products
- Consists of fully assembled, empty AAV9 capsids
- Represents serotype-specific conformational epitopes
- Reliable positive control for ELISA
- Fully assembled, empty AAV9 capsids
- Serotype-specific conformational epitopes
- Reliable positive control for dot blot, western blot, ELISA
- Quality controlled for purity, AAV titer and filling grade
- 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.
- Fully assembled AAV9 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.
- ELISA for quantitation of AAV9 capsids
- Detection of full and empty AAV9 capsids
- ADK9 antibody is used as capture and detection antibody
- get results in less than 2 hours and increase your daily sample throughput
- easy workflow & process integration
- same accuracy and reproducibility as the standard AAV9 Titration ELISA
- ADK9 antibody is used as capture and detection antibody
- Mouse monoclonal
- Suitable for ELISA, ICC/IF, IP and WB
- Reacts with VP1 of AAV1, AAV2, AAV3, AAV5, AAV6, AAV7, AAV8, AAV9 and AAVDJ
- Isotype: IgG2a
- Mouse monoclonal
- Suitable for affinity chromatography, dot blot, ICC/IF, IP and WB
- Reacts with denatured VP1, VP2, VP3 proteins of AAV1, 2, 3, 5, 6, 7, 8, 9, rh10, DJ
- Isotype: IgG1
- Rabbit polyclonal
- Suitable for ELISA, ICC/IF, IP and WB
- Reacts with AAV1, AAV2, AAV3, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh10, AAVDJ and AAVrh74, weakly with AAV4
- Strong affinity for denatured capsid proteins
- Weak affinity for native capsids
- Purified, liquid
- Mouse monoclonal
- Suitable for dot blot, ELISA, ICC and neutralization assay
- Reacts with AAV9 intact particles
- Isotype: IgA kappa
- Purified, lyophilized
- Recombinant antibody
- Suitable for dot blot, ELISA and neutralization assay
- Reacts with AAV9 intacts particles
- Isotype: mouse IgG1
- Recombinant chimeric (human Fc region)
- Suitable for dot blot, neutralization assay and serology ELISA
- Reacts with AAV9 intact capsids
- Isotype: IgG1
- Rapid lateral flow test for the detection of AAV9 capsids
- Semi-quantitative AAV titer determination in cell lysates and purified preparations
- Detection of full and empty AAV9 capsids
- Highly effective density gradient media for the separation of full and empty AAV particles
- Designed for the purification of viruses (such as AAV)
- Useful to isolate different cell types and organelles
- Ready-made, sterile and endotoxin-tested solution of iodixanol