All lanes : Anti-SARS2-CoV-2 Protein 3a 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
Lane 2 : HEK-293T transfected with SARS-CoV-2 Protein 3a expression vector containing a myc-His-tag®, whole cell lysate
Lane 3 : HEK-293T transfected with SARS-CoV-1 Protein 3a 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 (Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated)

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

Immunohistochemical analysis of paraffin-embedded HEK-293T cells labelling SARS2-CoV-2 Protein 3a 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 Protein 3a expression vector containing a myc-His-tag® Panel B: HEK-293T transfected with SARS-CoV-1 Protein 3a vector containing a myc-His-tag®. Panel C: Incubate HEK-293T transfected with SARS-CoV-2 Protein 3a 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.

Immunohistochemical analysis of paraffin-embedded Human lung tissue labelling SARS2-CoV-2 Protein 3a 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 ab280952 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.

Immunohistochemical analysis of paraffin-embedded Mouse lung tissue labelling SARS2-CoV-2 Protein 3a 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 ab280952 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.

Immunohistochemical analysis of paraffin-embedded Rat lung tissue labelling SARS2-CoV-2 Protein 3a 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 ab280952 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.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized 293T cells labelling SARS2-CoV-2 Protein 3a 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 Protein 3a expression vector, and no staining in 293T cells transfected with myc-tagged SARS-COV-1 Protein 3a 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.

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized HEK-293T (human embryonic kidney) transfected with myc-tagged SARS-CoV-2 Protein 3a expression construct (Right) with ab280953 at 1/500 dilution compared with HEK-293T (human embryonic kidney) transfected with myc-tagged SARS-COV-1 Protein 3a expression construct (Left). 

A Goat anti rabbit IgG (Alexa Fluor® 488, ab150077 at 1/2000 dilution was used as the secondary antibody.

SARS-CoV-2 Protein 3a was immunoprecipitated from 0.35 mg HEK-293T (human embryonic kidney) transfected with SARS-CoV-2 Protein 3a 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 Protein 3a expression vector containing a myc-His-tag®, whole cell lysate 20 μg

Lane 2: ab280953 IP in HEK-293T transfected with SARS-CoV-2 Protein 3a 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 Protein 3a expression vector containing a myc-His-tag® whole cell lysate

Blocking and dilution buffer and concentration: 5% NFDM/TBST.

Exposure time: 3 seconds.