Recombinant Rabbit monoclonal [EPR24848-9] to SARS-CoV2 orf3a (ab280953)
Key features and details
- Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
- Rabbit monoclonal [EPR24848-9] to SARS CoV-2 ORF3a Protein
- Suitable for: Flow Cyt (Intra), ICC/IF, WB, IP, IHC-P, ELISA
Related conjugates and formulations
Overview
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Product name
Rabbit monoclonal [EPR24848-9] to SARS-CoV2 orf3a
See all SARS CoV-2 ORF3a Protein primary antibodies -
Description
Rabbit monoclonal [EPR24848-9] to SARS CoV-2 ORF3a Protein -
Host species
Rabbit -
Tested applications
Suitable for: Flow Cyt (Intra), ICC/IF, WB, IP, IHC-P, ELISAmore details -
Species reactivity
Reacts with: SARS-CoV-2 -
Immunogen
Recombinant fragment. This information is proprietary to Abcam and/or its suppliers.
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Positive control
- WB: HEK-293T cells transfected with myc-tagged SARS-CoV-2 ORF3a Protein. IHC-P: 293T cells transfected with myc-tagged SARS-CoV-2 ORF3a Protein. ICC/IF: 293T cells transfected with myc-tagged SARS-CoV-2 ORF3a Protein. Flow Cyt(Intra): 293T cells transfected with myc-tagged SARS-CoV-2 ORF3a Protein. IP: 293T cells transfected with myc-tagged SARS-CoV-2 ORF3a Protein.
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General notes
This product is a recombinant monoclonal antibody, which offers several advantages including:
- - High batch-to-batch consistency and reproducibility
- - Improved sensitivity and specificity
- - Long-term security of supply
- - Animal-free production
Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb® patents.
Properties
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Form
Liquid -
Storage instructions
Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C long term. Avoid freeze / thaw cycle. -
Storage buffer
pH: 7.2
Preservative: 0.01% Sodium azide
Constituents: 59.94% PBS, 40% Glycerol (glycerin, glycerine), 0.05% BSA -
Concentration information loading...
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Purity
Protein A purified -
Clonality
Monoclonal -
Clone number
EPR24848-9 -
Isotype
IgG
Associated products
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Alternative Versions
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Compatible Secondaries
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Isotype control
Applications
The Abpromise guarantee
Our Abpromise guarantee covers the use of ab280953 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Application | Abreviews | Notes |
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Flow Cyt (Intra) |
1/500.
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ICC/IF | (2) |
1/500.
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WB | (2) |
1/1000.
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IP |
1/1000.
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IHC-P |
1/1000. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
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ELISA |
Use at an assay dependent concentration.
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Notes |
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Flow Cyt (Intra)
1/500. |
ICC/IF
1/500. |
WB
1/1000. |
IP
1/1000. |
IHC-P
1/1000. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol. |
ELISA
Use at an assay dependent concentration. |
Target
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Relevance
ORF3a is the largest accessory protein in SARS-CoV-2 virus, containing 274 amino acids. ORF3a is involved in viral entry within the host and has immunogenic properties. Moreover, it is involved in pro-inflammatory cytokine and chemokine production by activating various signal transduction pathways, leading to cytokine storm Covid-19 patients. ORF3a is also implicated in ion channel formation and modulates release of virus from the host cell. -
Alternative names
- SARS CoV-2 Protein 3a antibody
Images
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All lanes : Rabbit monoclonal [EPR24848-9] to SARS-CoV2 orf3a (ab280953) at 1/1000 dilution
Lanes 1 & 5 : Expi293 transfected with SARS-CoV-2 ORF3a protein expression vector (His-tagged) cell lysate at 40 µg
Lanes 2 & 6 : Expi293 transfected with SARS-CoV-2 ORF3a protein expression vector (His-tagged) cell lysate at 20 µg
Lanes 3 & 7 : Expi293 transfected with SARS-CoV-1 ORF3a protein expression vector (His-tagged) cell lysate at 40 µg
Lanes 4 & 8 : Expi293 transfected with SARS-CoV-1 ORF3a protein expression vector (His-tagged) cell lysate at 20 µg
Observed band size: 26 kDa why is the actual band size different from the predicted?Lanes 1 – 4: Green – ab280953 observed at 26 kDa.
Lanes 5 – 8: Red – loading control ab18184 (Mouse monoclonal [HIS.H8] to 6X His tag®) observed at 26 kDa.
ab280953 was shown to bind specifically to SARS CoV-2 ORF3a Protein in Western blot. Samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (ab216776) at 1/20000 dilution.
Blocking buffer: 3% milk in TBS-0.1 % Tween® 20 (TBS-T)
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All lanes : Rabbit monoclonal [EPR24848-9] to SARS-CoV2 orf3a (ab280953) at 1/1000 dilution
Lane 1 : HEK-293T (human embryonic kidney) transfected with an empty vector (vector control) containing a myc-His-tag®, whole cell lysate
Lane 2 : HEK-293T transfected with SARS-CoV-2 ORF3a Protein expression vector containing a myc-His-tag®, whole cell lysate
Lane 3 : HEK-293T transfected with SARS-CoV-1 ORF3a Protein expression vector containing a myc-His-tag®, whole cell lysate
Lysates/proteins at 20 µg per lane.
Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution
Observed band size: 23 kDa why is the actual band size different from the predicted?Blocking and Diluting buffer and concentration: 5% NFDM/TBST
Exposure time 5.5 seconds
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SARS CoV-2 ORF3a Protein was immunoprecipitated from 0.35 mg HEK-293T (human embryonic kidney) transfected with SARS CoV-2 ORF3a Protein expression vector containing a myc-His-tag®, whole cell lysate 20 µg with ab280953 at 1/30 dilution (2µg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab280953 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/5000 dilution.
Lane 1: HEK-293T (human embryonic kidney) transfected with SARS CoV-2 ORF3a Protein expression vector containing a myc-His-tag®, whole cell lysate 20 μg
Lane 2: ab280953 IP in HEK-293T transfected with SARS CoV-2 ORF3a Protein expression vector containing a myc-His-tag® whole cell lysate
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab280953 in HEK-293T transfected with SARS CoV-2 ORF3a Protein expression vector containing a myc-His-tag® whole cell lysate
Blocking and dilution buffer and concentration: 5% NFDM/TBST.
Exposure time: 3 seconds.
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Immunohistochemical analysis of paraffin-embedded HEK-293T cells labelling SARS CoV-2 ORF3a Protein with ab280953 at 1/1000 (0.53 μg/ml) dilution followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Panel A : HEK-293T (human epithelial cell line from embryonic kidney transformed with large T antigen) transfected with SARS CoV-2 ORF3a Protein expression vector containing a myc-His-tag® Panel B: HEK-293T transfected with SARS-CoV-1 ORF3a Protein vector containing a myc-His-tag®. Panel C: Incubate HEK-293T transfected with SARS CoV-2 ORF3a Protein expression vector containing a myc-His-tag® with secondary antibody only, but not ab280953. Panel D: HEK-293T transfected with control vector containing a myc-His-tag®. Positive staining on (A) HEK-293T transfected with SARS-CoV-2 Protein 3a expression vector. No staining on (B) HEK-293T transfected with a SARS-CoV-1 Protein 3a expression vector, (C) HEK-293T transfected with SARS-CoV-2 Protein 3a expression vector and incubated with secondary antibody only and (D) HEK-293T transfected with empty vector. The section was incubated with ab280953 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin.
Secondary antibody only control: Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used.
Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.
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Immunohistochemical analysis of paraffin-embedded Human lung tissue labelling SARS CoV-2 ORF3a Protein with ab280953 at 1/1000 (0.53 μg/ml) dilution followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). No staining on human lung. The section was incubated with ab280953 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin.
Secondary antibody only control: Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used.
Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.
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Immunohistochemical analysis of paraffin-embedded Mouse lung tissue labelling SARS CoV-2 ORF3a Protein with ab280953 at 1/1000 (0.53 μg/ml) dilution followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). No staining on mouse lung. The section was incubated with ab280953 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin.
Secondary antibody only control: Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used.
Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.
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Immunohistochemical analysis of paraffin-embedded Rat lung tissue labelling SARS CoV-2 ORF3a Protein with ab280953 at 1/1000 (0.53 μg/ml) dilution followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). No staining on rat lung. The section was incubated with ab280953 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin.
Secondary antibody only control: Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used.
Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.
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Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized 293T cells labelling SARS CoV-2 ORF3a Protein with ab280953 at 1/500 dilution (1.06 μg/ml), followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) antibody at 1/1000 dilution (Green). Confocal image showing cytoplasmic staining in 293T cells transfected with myc-tagged SARS CoV-2 ORF3a Protein expression vector, and no staining in 293T cells transfected with myc-tagged SARS-COV-1 ORF3a protein expression vector. Myc-Tag Mouse mAb (Alexa Fluor® 647) was used to counterstain myc-tag at 1/100 dilution (Red). The Nuclear counterstain was DAPI (Blue).
Secondary antibody only control: Secondary antibody is ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) at 1/1000 dilution.
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Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized HEK-293T (human embryonic kidney) transfected with myc-tagged SARS CoV-2 ORF3a Protein expression construct (Right) with ab280953 at 1/500 dilution compared with HEK-293T (human embryonic kidney) transfected with myc-tagged SARS-COV-1 ORF3a Protein expression construct (Left).
A Goat anti rabbit IgG (Alexa Fluor® 488, ab150077 at 1/2000 dilution was used as the secondary antibody.
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Recombinant Anti-SARS CoV-2 ORF3a Protein antibody [EPR24848-9] (ab280953)
Plates were coated with a recombinant protein ORF3 at 1000 ng/ml.
Primary antibody (ab280953) concentration range 0.045-100 ng /ml.
Pre-adsorbed secondary antibody, goat anti-rabbit IgG H&L (HRP, ab97080) used at 1/2000 dilution.
Protocols
To our knowledge, customised protocols are not required for this product. Please try the standard protocols listed below and let us know how you get on.
Datasheets and documents
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SDS download
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Datasheet download
References (1)
ab280953 has been referenced in 1 publication.
- Zhang X et al. SARS-CoV-2 ORF3a induces RETREG1/FAM134B-dependent reticulophagy and triggers sequential ER stress and inflammatory responses during SARS-CoV-2 infection. Autophagy 18:2576-2592 (2022). PubMed: 35239449