Anti-SARS-CoV2 orf3a antibody [EPR24848-9]

9 product images

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  Immunoprecipitation - Anti-SARS-CoV2 orf3a antibody [EPR24848-9] (ab280953)

  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)(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 (Rabbit IgG, monoclonal [EPR25A] - Isotype Control 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.

  All lanes: Immunoprecipitation - Anti-SARS-CoV2 orf3a antibody [EPR24848-9] (ab280953)

  Observed band size: 23 kDa

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  Western blot - Anti-SARS-CoV2 orf3a antibody [EPR24848-9] (ab280953)

  SARS-CoV2 orf3a Western blot staining using rabbit Anti-SARS-CoV2 orf3a antibody

  Lanes 1 – 4: Green – ab280953 observed at 26 kDa.
  

  Lanes 5 – 8: Red – loading control Anti-6X His tag® antibody [HIS.H8] 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 (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) at 1/20000 dilution.

  Blocking buffer: 3% milk in TBS-0.1 % Tween® 20 (TBS-T)

  All lanes: Western blot - Anti-SARS-CoV2 orf3a antibody [EPR24848-9] (ab280953) at 1/1000 dilution

  Lanes 1 and 5: Expi293 transfected with SARS-CoV-2 ORF3a protein expression vector (His-tagged) cell lysate at 40 µg

  Lanes 2 and 6: Expi293 transfected with SARS-CoV-2 ORF3a protein expression vector (His-tagged) cell lysate at 20 µg

  Lanes 3 and 7: Expi293 transfected with SARS-CoV-1 ORF3a protein expression vector (His-tagged) cell lysate at 40 µg

  Lanes 4 and 8: Expi293 transfected with SARS-CoV-1 ORF3a protein expression vector (His-tagged) cell lysate at 20 µg

  Observed band size: 26 kDa

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  Western blot - Anti-SARS-CoV2 orf3a antibody [EPR24848-9] (ab280953)

  SARS-CoV2 orf3a Western blot staining using rabbit Anti-SARS-CoV2 orf3a antibody

  Blocking and Diluting buffer and concentration: 5% NFDM/TBST
  

  Exposure time 5.5 seconds

  All lanes: Western blot - Anti-SARS-CoV2 orf3a antibody [EPR24848-9] (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 at 20 µg

  Lane 2: HEK-293T transfected with SARS-CoV-2 ORF3a Protein expression vector containing a myc-His-tag®, whole cell lysate at 20 µg

  Lane 3: HEK-293T transfected with SARS-CoV-1 ORF3a Protein expression vector containing a myc-His-tag®, whole cell lysate at 20 µg

  Secondary

  All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/100000 dilution

  Observed band size: 23 kDa

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SARS-CoV2 orf3a antibody [EPR24848-9] (ab280953)

  SARS-CoV2 orf3a Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) staining using rabbit Anti-SARS-CoV2 orf3a antibody

  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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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SARS-CoV2 orf3a antibody [EPR24848-9] (ab280953)

  SARS-CoV2 orf3a Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) staining using rabbit Anti-SARS-CoV2 orf3a antibody

  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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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SARS-CoV2 orf3a antibody [EPR24848-9] (ab280953)

  SARS-CoV2 orf3a Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) staining using rabbit Anti-SARS-CoV2 orf3a antibody

  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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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SARS-CoV2 orf3a antibody [EPR24848-9] (ab280953)

  SARS-CoV2 orf3a Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) staining using rabbit Anti-SARS-CoV2 orf3a antibody

  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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  Flow Cytometry (Intracellular) - Anti-SARS-CoV2 orf3a antibody [EPR24848-9] (ab280953)

  SARS-CoV2 orf3a Flow Cytometry (Intracellular) staining using rabbit Anti-SARS-CoV2 orf3a antibody

  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, Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077 at 1/2000 dilution was used as the secondary antibody.

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  ELISA - Anti-SARS-CoV2 orf3a antibody [EPR24848-9] (ab280953)

  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, Goat Anti-Rabbit IgG H&L (HRP) preadsorbed ab97080) used at 1/2000 dilution.