AAV2 VP1 + VP2 + VP3, recombinant proteins, set
Western blot analysis of recombinant AAV2 capsid proteins (Cat. No. 640823, 640824, 640825) with fluorescent B1 antibody (Cat. No. 61058-488). Western blot analysis was performed on the precise molar ratio of 1:1:10 (VP1:VP2:VP3) either in separate lanes or combined in one lane. The PVDF membranes were blocked with 5% dry milk in PBST (PBS + 0.1% Tween 20) for 1 h at RT. The primary antibody anti-AAV VP1/VP2/VP3, B1 AFDyeTM 488 (Cat. No. 61058-488) was diluted in blocking buffer (antibody concentration 130 ng/ml) and incubated for 1 h at RT. The bands were visualized by fluorescent detection.
Western blot analysis of recombinant AAV2 capsid proteins (Cat. No. 640823, 640824, 640825) with B1 antibody (Cat. No. 690058). Western blot analysis was performed on the precise molar ratio of 1:1:10 (VP1:VP2:VP3) either in separate lanes or combined in one lane The PVDF membranes were blocked with 5% dry milk in PBST (PBS + 0.1% Tween 20) for 1 h at RT. The primary antibody anti-AAV VP1/VP2/VP3 mouse monoclonal, B1 (Cat. No. 690058) was diluted in blocking buffer (antibody concentration 0.5 µg/ml) and incubated for 1 h at RT. The secondary antibody goat anti-mouse IgG HRP was also diluted in blocking buffer (antibody concentration 0.2 µg/ml) and incubated for 1 h at RT. The bands were visualized by chemiluminescent detection using PierceTM ECL Western Blotting Substrate.
Western blot analysis of recombinant AAV2 capsid proteins (Cat. No. 640823, 640824, 640825) and denatured AAV2 cpasids with B1 antibody (Cat. No. 690058). Western blot analysis was performed on 3.3E+09 denatured AAV2 capsids and recombinant AAV2 VP proteins (ratio 1:1:10 - VP1:VP2:VP3). The PVDF membranes were blocked with 5% dry milk in PBST (PBS + 0.1% Tween 20) for 1 h at RT than probed with 0.2 µg/ml (AAV2 capsids) or 0.5 µg/ml (recombinant VP proteins) anti-AAV VP1/VP2/VP3 mouse monoclonal, B1 as primary antibody diluted in blocking buffer for 1 h at 4°C. The secondary antibody goat anti-mouse IgG HRP was also diluted in blocking buffer (antibody concentration 0.2 µg/ml) and incubated for 1 h at RT. The bands were visualized by chemiluminescent detection using PierceTM ECL Western Blotting Substrate.
Analysis of AAV2 VP1 + VP2 + VP3, recombinant proteins (Cat. No. 72001) with various stoichiometric ratios by Simple WesternTM, a CE-immunoassay platform from ProteinSimple, a Bio-Techne brand. Capsid proteins were detected using an anti-AAV VP1/VP2/VP3 mouse monoclonal antibody (Cat. No. 690058).
Western blot analysis of recombinant AAV2 capsid proteins (Cat. No. 640823, 640824, 640825) with B1 antibody (Cat. No. 690058).
Western blot analysis was performed on different molar ratios of VP1:VP2:VP3 (1:1:10, 2:2:5, 1:1:5, 3:3:10 and 3:3:3) compared to 1E+10 denatured AAV2 capsids. The capsids were denatured for 10 min at 95°C. The PVDF membrane was blocked with 5% dry milk in PBST (PBS + 0.1% Tween 20) for 1 h at RT. The primary antibody anti-AAV VP1/VP2/VP3 mouse monoclonal, B1 (Cat. No. 690058) was diluted in blocking buffer (antibody concentration 0.5 µg/ml) and incubated for 1 h at RT. The secondary antibody goat anti-mouse IgG HRP was also diluted in blocking buffer (antibody concentration 0.2 µg/ml) and incubated for 1 h at RT. The bands were visualized by chemiluminescent detection using PierceTM ECL Western Blotting Substrate.
Please note that various amounts of total protein were loaded in each lane.
Western blot analysis was performed on different molar ratios of VP1:VP2:VP3 (1:1:10, 2:2:5, 1:1:5, 3:3:10 and 3:3:3) compared to 1E+10 denatured AAV2 capsids. The capsids were denatured for 10 min at 95°C. The PVDF membrane was blocked with 5% dry milk in PBST (PBS + 0.1% Tween 20) for 1 h at RT. The primary antibody anti-AAV VP1/VP2/VP3 mouse monoclonal, B1 (Cat. No. 690058) was diluted in blocking buffer (antibody concentration 0.5 µg/ml) and incubated for 1 h at RT. The secondary antibody goat anti-mouse IgG HRP was also diluted in blocking buffer (antibody concentration 0.2 µg/ml) and incubated for 1 h at RT. The bands were visualized by chemiluminescent detection using PierceTM ECL Western Blotting Substrate.
Please note that various amounts of total protein were loaded in each lane.
Pipetting scheme for western blot analysis using the AAV2 capsid proteins (Cat. No. 640823, 640824, 640825) in separate lanes. To analyze the molar ratio of 1:1:10, it is recommended to load VP1, VP2 and VP3 as described in the pipetting scheme above. Therefore, the indicated volumes of the proteins (concentration 100 µg/ml) should be diluted with the appropriate amount of sample buffer and distilled water.
10 µl of each solution can be separately loaded onto the SDS PAGE and analyzed by Western blot using the B1 antibody (Cat. No. 690058, Cat. No. 61058-488, Cat. No. 61058-647).
10 µl of each solution can be separately loaded onto the SDS PAGE and analyzed by Western blot using the B1 antibody (Cat. No. 690058, Cat. No. 61058-488, Cat. No. 61058-647).
Pipetting scheme for western blot analysis using a mix of the AAV2 capsid proteins (Cat. No. 640823, 640824, 640825). To create a VP mixture with the molar ratio 1:1:10 (VP1:VP2:VP3), please pre-dilute VP1 and VP2 1:10 to yield a final concentration of 10 µg/ml (green table). Pipette the pre-diluted VP1 and VP2 proteins and mix them with the undiluted VP3 protein in your sample buffer and water (blue table). The example with 2x and 3x sample buffer and the required volumes are indicated in the pipetting scheme. Thus, in one lane, 10 µl of the VP mix can be loaded onto the SDS PAGE and analyzed by Western blot using the B1 antibody (Cat. No. 690058, Cat. No. 61058-488, Cat. No. 61058-647).
Undiluted = 100 µg/ml, pre-diluted = 10 µg/ml
Undiluted = 100 µg/ml, pre-diluted = 10 µg/ml
SDS PAGE analysis to evaluate the purity of the AAV2 VP1, VP2 and VP3 (Cat. No. 72001). To perform SDS PAGE analysis, 1 µg of each AAV2 VP protein was diluted in 10 µl PBS and sample buffer and denatured at 95°C for 10 min. The samples were loaded onto a 4-20% gradient gel (40 min at 200 V). Afterwards, the gel was stained for 1 h at RT with Coomassie solution and destained with water. The purity of AAV2 VP1, VP2 and VP3 is > 90%.
Western blot analysis of recombinant AAV2 capsid proteins (Cat. No. 640823, 640824, 640825) with fluorescent B1 antibody (Cat. No. 61058-488). Western blot analysis was performed on the precise molar ratio of 1:1:10 (VP1:VP2:VP3) either in separate lanes or combined in one lane. The PVDF membranes were blocked with 5% dry milk in PBST (PBS + 0.1% Tween 20) for 1 h at RT. The primary antibody anti-AAV VP1/VP2/VP3, B1 AFDyeTM 488 (Cat. No. 61058-488) was diluted in blocking buffer (antibody concentration 130 ng/ml) and incubated for 1 h at RT. The bands were visualized by fluorescent detection.
Western blot analysis of recombinant AAV2 capsid proteins (Cat. No. 640823, 640824, 640825) with B1 antibody (Cat. No. 690058). Western blot analysis was performed on the precise molar ratio of 1:1:10 (VP1:VP2:VP3) either in separate lanes or combined in one lane The PVDF membranes were blocked with 5% dry milk in PBST (PBS + 0.1% Tween 20) for 1 h at RT. The primary antibody anti-AAV VP1/VP2/VP3 mouse monoclonal, B1 (Cat. No. 690058) was diluted in blocking buffer (antibody concentration 0.5 µg/ml) and incubated for 1 h at RT. The secondary antibody goat anti-mouse IgG HRP was also diluted in blocking buffer (antibody concentration 0.2 µg/ml) and incubated for 1 h at RT. The bands were visualized by chemiluminescent detection using PierceTM ECL Western Blotting Substrate.
Western blot analysis of recombinant AAV2 capsid proteins (Cat. No. 640823, 640824, 640825) and denatured AAV2 cpasids with B1 antibody (Cat. No. 690058). Western blot analysis was performed on 3.3E+09 denatured AAV2 capsids and recombinant AAV2 VP proteins (ratio 1:1:10 - VP1:VP2:VP3). The PVDF membranes were blocked with 5% dry milk in PBST (PBS + 0.1% Tween 20) for 1 h at RT than probed with 0.2 µg/ml (AAV2 capsids) or 0.5 µg/ml (recombinant VP proteins) anti-AAV VP1/VP2/VP3 mouse monoclonal, B1 as primary antibody diluted in blocking buffer for 1 h at 4°C. The secondary antibody goat anti-mouse IgG HRP was also diluted in blocking buffer (antibody concentration 0.2 µg/ml) and incubated for 1 h at RT. The bands were visualized by chemiluminescent detection using PierceTM ECL Western Blotting Substrate.
Analysis of AAV2 VP1 + VP2 + VP3, recombinant proteins (Cat. No. 72001) with various stoichiometric ratios by Simple WesternTM, a CE-immunoassay platform from ProteinSimple, a Bio-Techne brand. Capsid proteins were detected using an anti-AAV VP1/VP2/VP3 mouse monoclonal antibody (Cat. No. 690058).
Western blot analysis of recombinant AAV2 capsid proteins (Cat. No. 640823, 640824, 640825) with B1 antibody (Cat. No. 690058).
Western blot analysis was performed on different molar ratios of VP1:VP2:VP3 (1:1:10, 2:2:5, 1:1:5, 3:3:10 and 3:3:3) compared to 1E+10 denatured AAV2 capsids. The capsids were denatured for 10 min at 95°C. The PVDF membrane was blocked with 5% dry milk in PBST (PBS + 0.1% Tween 20) for 1 h at RT. The primary antibody anti-AAV VP1/VP2/VP3 mouse monoclonal, B1 (Cat. No. 690058) was diluted in blocking buffer (antibody concentration 0.5 µg/ml) and incubated for 1 h at RT. The secondary antibody goat anti-mouse IgG HRP was also diluted in blocking buffer (antibody concentration 0.2 µg/ml) and incubated for 1 h at RT. The bands were visualized by chemiluminescent detection using PierceTM ECL Western Blotting Substrate.
Please note that various amounts of total protein were loaded in each lane.
Western blot analysis was performed on different molar ratios of VP1:VP2:VP3 (1:1:10, 2:2:5, 1:1:5, 3:3:10 and 3:3:3) compared to 1E+10 denatured AAV2 capsids. The capsids were denatured for 10 min at 95°C. The PVDF membrane was blocked with 5% dry milk in PBST (PBS + 0.1% Tween 20) for 1 h at RT. The primary antibody anti-AAV VP1/VP2/VP3 mouse monoclonal, B1 (Cat. No. 690058) was diluted in blocking buffer (antibody concentration 0.5 µg/ml) and incubated for 1 h at RT. The secondary antibody goat anti-mouse IgG HRP was also diluted in blocking buffer (antibody concentration 0.2 µg/ml) and incubated for 1 h at RT. The bands were visualized by chemiluminescent detection using PierceTM ECL Western Blotting Substrate.
Please note that various amounts of total protein were loaded in each lane.
Pipetting scheme for western blot analysis using the AAV2 capsid proteins (Cat. No. 640823, 640824, 640825) in separate lanes. To analyze the molar ratio of 1:1:10, it is recommended to load VP1, VP2 and VP3 as described in the pipetting scheme above. Therefore, the indicated volumes of the proteins (concentration 100 µg/ml) should be diluted with the appropriate amount of sample buffer and distilled water.
10 µl of each solution can be separately loaded onto the SDS PAGE and analyzed by Western blot using the B1 antibody (Cat. No. 690058, Cat. No. 61058-488, Cat. No. 61058-647).
10 µl of each solution can be separately loaded onto the SDS PAGE and analyzed by Western blot using the B1 antibody (Cat. No. 690058, Cat. No. 61058-488, Cat. No. 61058-647).
Pipetting scheme for western blot analysis using a mix of the AAV2 capsid proteins (Cat. No. 640823, 640824, 640825). To create a VP mixture with the molar ratio 1:1:10 (VP1:VP2:VP3), please pre-dilute VP1 and VP2 1:10 to yield a final concentration of 10 µg/ml (green table). Pipette the pre-diluted VP1 and VP2 proteins and mix them with the undiluted VP3 protein in your sample buffer and water (blue table). The example with 2x and 3x sample buffer and the required volumes are indicated in the pipetting scheme. Thus, in one lane, 10 µl of the VP mix can be loaded onto the SDS PAGE and analyzed by Western blot using the B1 antibody (Cat. No. 690058, Cat. No. 61058-488, Cat. No. 61058-647).
Undiluted = 100 µg/ml, pre-diluted = 10 µg/ml
Undiluted = 100 µg/ml, pre-diluted = 10 µg/ml
SDS PAGE analysis to evaluate the purity of the AAV2 VP1, VP2 and VP3 (Cat. No. 72001). To perform SDS PAGE analysis, 1 µg of each AAV2 VP protein was diluted in 10 µl PBS and sample buffer and denatured at 95°C for 10 min. The samples were loaded onto a 4-20% gradient gel (40 min at 200 V). Afterwards, the gel was stained for 1 h at RT with Coomassie solution and destained with water. The purity of AAV2 VP1, VP2 and VP3 is > 90%.
Key Features
• Recombinant AAV2 VP1-3 capsid protein set
• Useful for ratio analysis
• Precise molar concentration
• Suitable for WB, dot blot, CE, SDS PAGE
• Useful for ratio analysis
• Precise molar concentration
• Suitable for WB, dot blot, CE, SDS PAGE
Not available in China and in the Russian Federation.
For US shipment, the packages are only sent out for delivery on Mondays and Tuesdays, in the EU from Monday until Wednesday.
Included products:
Product description
| Quantity | 10 µg each protein |
|---|---|
| Application | Capillary electrophoresis (CE), Dot blot, SDS PAGE, WB |
| Purification | Ni-NTA chromatography |
| Storage | -80°C |
| Intended use | Research use only |
| Concentration | 100 µg/ml (VP1: 1.19 µM, VP2: 1.45 µM, VP3: 1.61 µM) |
| Formulation | Liquid, 6 M urea in PBS |
| Source | Escherichia coli |
| Molecular weight | VP1: 84.1 kDa, VP2: 68.9 kDa, VP3: 62.2 kDa (calculated Mw from aa sequence) |
| Purity | > 90% (determined by SDS PAGE) |
| Product description | N-terminal His-tagged (MGSSHHHHHHSSGLVPRGSH) recombinant AAV2 capsid proteins VP1 + VP2 + VP3 |
Applications
| Tested applications | Tested dilutions |
|---|---|
| Capillary electrophoresis (CE) | Assay dependent |
| Dot Blot | 100 ng, depending on primary antibody and detection method |
| SDS PAGE | 1 µg |
| Western Blot (WB) | 5-20 ng, depending on primary antibody and detection method |
Background
The AAV capsid consists of three capsid proteins, i.e. VP1, VP2 and VP3, which differ in their N-terminus and encapsulate the genomic ssDNA. In native virus particles, the three proteins form subunits with a ratio of 1:1:10 (VP1:VP2:VP3), in a total number of 60 subunits per capsid.
This set of recombinant AAV2 VP1, VP2 and VP3 can be used to create a mixture with the precise molar ratio of 1:1:10 to compare the protein composition of the viral capsid in your sample by protein detection methods, e.g. western blot.
All three recombinant AAV2 capsid proteins are available as set (Cat. No. 72001) or as individual proteins (Cat. No. 640823, 640824, 640825).
Note: please find an example how to prepare western blot samples in the pipetting scheme below. Aliquots of the remaining samples can be stored at -80°C for reuse.
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