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

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SARS-CoV2 orf3a antibody [EPR24848-9] (AB280953)

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.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-SARS-CoV2 orf3a antibody [EPR24848-9] (AB280953)

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.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SARS-CoV2 orf3a antibody [EPR24848-9] (AB280953)

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.

• IP

Supplier Data

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)(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.

All lanes:

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

Observed band size: 23 kDa

false

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SARS-CoV2 orf3a antibody [EPR24848-9] (AB280953)

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.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SARS-CoV2 orf3a antibody [EPR24848-9] (AB280953)

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.

• ELISA

Lab

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, ab97080) used at 1/2000 dilution.

• WB

Lab

Western blot - Anti-SARS-CoV2 orf3a antibody [EPR24848-9] (AB280953)

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)

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

false

• WB

Supplier Data

Western blot - Anti-SARS-CoV2 orf3a antibody [EPR24848-9] (AB280953)

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) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Observed band size: 23 kDa

false