All lanes : Anti-SARS-CoV-2 nucleocapsid protein antibody [EPR24334-118] (ab271180) at 1/1000 dilution

Lane 1 : HEK-293T (human embryonic kidney) transfected with SARS-CoV-2 Nucleocapsid protein expression vector containing a myc-His-tag®, whole cell lysate
Lane 2 : HEK-293T (human embryonic kidney) transfected with SARS-CoV-1 Nucleocapsid protein expression vector containing a myc-His-tag®, whole cell lysate
Lane 3 : HEK-293T (human embryonic kidney) transfected with MERS Nucleocapsid proteinexpression vector containing a myc-His-tag®, whole cell lysate

Lysates/proteins at 10 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: 51 kDa why is the actual band size different from the predicted?

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

Exposure time 7.75 seconds

All lanes : Anti-SARS-CoV-2 nucleocapsid protein antibody [EPR24334-118] (ab271180) at 1/1000 dilution

Lane 1 : SARS-CoV-2 Nucleocapsid Recombinant Protein at 0.1 µg
Lane 2 : SARS-CoV-2 infected Vero for 48 hours
Lane 3 : Uninfected Vero

Observed band size: 51 kDa why is the actual band size different from the predicted?

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

Data was kindly provided by Dr. Hangping Yao, State Key Laboratory for Diagnosis and Treatment of Infectious Diseases in Zhejiang University, School of Medicine.

Immunohistochemical analysis of paraffin-embedded HEK-293T (human epithelial cell line from embryonic kidney transformed with large T antigen) transfected with SARS-CoV-2 Nucleocapsid protein expression vector containing a myc-His-tag® (Panel A) 
HEK-293T transfected with SARS-CoV-1 Nucleocapsid protein expression vector containing a myc-His-tag® (Panel B) HEK-293T transfected with MERS Nucleocapsid protein expression vector containing a myc-His-tag® (Panel C) HEK-293T transfected with control vector containing a myc-His-tag® (Panel D) with 271180 at 1/4000 dilution followed by ready to use Goat Anti-Rabbit IgG H&L (HRP polymer). Positive staining on (A) HEK-293T transfected with a SARS-CoV-2 Nucleocapsid protein expression vector is observed. No staining on (B) HEK-293T transfected with a SARS-CoV-1 Nucleocapsid protein expression vector, (C) HEK-293T transfected with MERS Nucleocapsid protein expression vector and (D) HEK-293T transfected with empty vector. The section was incubated with ab271180 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin.

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins

Immunohistochemical analysis of paraffin-embedded Human lung labeling SARS-CoV-2 with ab271180 at 1/4000 dilution followed by ready to use Goat Anti-Rabbit IgG H&L (HRP polymer). Negative control: No staining on human lung. The section was incubated with ab271180 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: Goat Anti-Rabbit IgG H&L (HRP polymer).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

Immunohistochemical analysis of paraffin-embedded Mouse lung labeling SARS-CoV-2 with ab271180 at 1/4000 dilution followed by ready to use Goat Anti-Rabbit IgG H&L (HRP polymer). Negative control: No staining on Mouse lung. The section was incubated with ab271180 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: Goat Anti-Rabbit IgG H&L (HRP polymer).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

Immunohistochemical analysis of paraffin-embedded Rat lung labeling SARS-CoV-2 with ab271180 at 1/4000 dilution followed by ready to use Goat Anti-Rabbit IgG H&L (HRP polymer). Negative control: No staining on Rat lung. The section was incubated with ab271180 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: Goat Anti-Rabbit IgG H&L (HRP polymer).

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 transfected with myc-tagged SARS-CoV-2 Nucleocapsid protein expression vector labelling SARS-CoV-2 with ab271180 at 1/1000 dilution, followed by ab150080 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 594) antibody at 1/1000 dilution (Red). ab202008 Anti-Myc tag mouse monoclonal antibody (Alexa Fluor® 488) was used to counterstain Myc tag at 1/200 dilution (Green). The nuclear counterstain was DAPI (Blue). Confocal image showing cytoplasmic staining in 293T cells transfected with myc-tagged SARS-CoV-2 Nucleocapsid protein expression vector.

 

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100-permeabilized Vero cells labelling SARS-CoV-2 with ab271180 at 1/1000 dilution, followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) antibody at 1/500 dilution. Fluorescent image showing positve staining in Vero cells after infecting with SARS-CoV-2 virus for 48 h. Nuclear counter stain DAPI. 
Two indenpendent optical fields were captured for imaging at each given antibody dilution.

This piece of data was kindly provided by Dr. Hangping Yao, State Key Laboratory for Diagnosis and Treatment of Infectious Diseases in Zhejiang University, School of Medicine

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100-permeabilized 293T cells transfected with myc-tagged SARS-CoV-1 and MERS Nucleocapsid protein labelling SARS-CoV-2 with ab271180 at 1/1000 dilution, followed by ab150080 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 594) antibody at 1/1000 dilution. ab202008 Anti-Myc tag mouse monoclonal antibody (Alexa Fluor® 488) was used to counterstain Myc tag at 1/200 dilution (Green). The nuclear counterstain was DAPI (Blue). Confocal image showing no staining in 293T cells transfected with myc-tagged SARS-CoV-1 and MERS Nucleocapsid protein, which are negative controls for SARS-CoV-2.

 

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized HEK-293T (human embryonic kidney) transfected with myc-tagged SARS-CoV-2 Nucleocapsid protein expression construct  labelling SARS-CoV-2 with ab271180 at 1/500 dilution (Right) compared with a Rabbit monoclonal IgG (ab172730) (Left) isotype control. 

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

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized HEK-293T (human embryonic kidney) transfected with myc-tagged SARS-CoV-1 Nucleocapsid protein expression construct (Left)/ HEK-293T (human embryonic kidney) transfected with myc-tagged MERS Nucleocapsid protein expression construct (Right). 

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

SARS-CoV-2 was immunoprecipitated from 0.35 mg HEK-293T (human embryonic kidney) transfected with SARS-CoV-2 Nucleocapsid protein expression vector containing a myc-His-tag®, whole cell lysate 5 µg with ab271180 at 1/30 dilution (2µg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab267373 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 Nucleocapsid protein expression vector containing a myc-His-tag® whole cell lysate 5 μg

Lane 2: ab271180 IP in HEK-293T transfected with SARS-CoV-2 Nucleocapsid protein expression vector containing a myc-His-tag® whole cell lysate.

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab271180 in HEK-293T transfected with SARS-CoV-2 Nucleocapsid protein expression vector containing a myc-His-tag® whole cell lysate

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

Exposure time: 3.25 seconds.

 

SARS-CoV-2 infected Vero cell supernatant was used for coating at 1/10 dilution. Primary antibody - ab271180 concentration range 5000~0 ng /ml. Secondary antibody Goat Anti-Rabbit IgG H&L (HRP) at 1/10000 dilution

Data was kindly provided by Dr. Hangping Yao, State Key Laboratory for Diagnosis and Treatment of Infectious Diseases in Zhejiang University, School of Medicine.

SARS-CoV-2 Nucleocapsid Recombinant Protein，SARS-CoV-1 Nucleocapsid Recombinant Protein，MERS Nucleocapsid Recombinant Protein at 1000 ng/ml concentration.

Primary antibody - ab271180 concentration range 1000~0 ng /ml. Secondary antibody Alkaline Phosphatase-conjugated AffiniPure Goat Anti-Rabbit IgG (H+L) at 1/2000 dilution.