Anti-SARS-CoV2 orf8 antibody [EPR24851-65] - BSA and Azide free

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-SARS-CoV2 orf8 antibody [EPR24851-65] - BSA and Azide free (AB284042)

Intracellular Flow Cytometry analysis of HEK-293T (human epithelial cell line from embryonic kidney transformed with large T antigen) cells transfected with a SARS-COV2 orf7a expression vector containing a GFP tag labeling SARS-CoV2 orf8 with ab283914 at 1/500 dilution (right). Cells were fixed with 4% Paraformaldehyde and permeabilised with 90% Methanol. A Goat anti-rabbit IgG (Alexa Fluor^® 647, ab150083) secondary antibody was used at 1/5000 dilution. Isotype control - Rabbit monoclonal IgG (left) (ab172730). No cross-reactivity with SARS-COV2 orf7a.

This data was developed using ab283914, the same antibody clone in a different buffer formulation.

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-SARS-CoV2 orf8 antibody [EPR24851-65] - BSA and Azide free (AB284042)

Intracellular Flow Cytometry analysis of HEK-293T (human epithelial cell line from embryonic kidney transformed with large T antigen) cells transfected with a SARS-CoV2 orf8 expression vector containing a myc tag labeling SARS-CoV2 orf8 with ab283914 at 1/500 dilution (right). Cells were fixed with 4% Paraformaldehyde and permeabilised with 90% Methanol. A Goat anti-rabbit IgG (Alexa Fluor^® 488, ab150081) secondary antibody was used at 1/5000 dilution. Isotype control - Rabbit monoclonal IgG (left) (ab172730).

This data was developed using ab283914, the same antibody clone in a different buffer formulation.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-SARS-CoV2 orf8 antibody [EPR24851-65] - BSA and Azide free (AB284042)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HEK-293T (human epithelial cell line from embryonic kidney transformed with large T antigen) cells labelling SARS-CoV2 orf8 with primary antibody anti-SARS-CoV2 orf8 (ab283914) at 1/100 dilution, followed by AlexaFluor^®594 Goat anti-Rabbit (green) (ab150088) secondary antibody at 1/1000 dilution. Confocal image showing cytoplasmic and nuclear staining in HEK-293T cells transfected with a SARS-CoV2 orf8 expression vector containing a myc tag, no staining in HEK-293T cells transfected with a SARS-COV2 orf7a expression vector containing a myc tag. Myc-Tag Mouse mab (Alexa Fluor^® 647) (red) was used to counterstain at 1/100 dilution. The nuclear counter stain is DAPI (blue).

This data was developed using ab283914, the same antibody clone in a different buffer formulation.

• IHC-P

AbReview84149****

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SARS-CoV2 orf8 antibody [EPR24851-65] - BSA and Azide free (AB284042)

This data was developed using ab283914, the same antibody clone in a different buffer formulation. ab283914 staining SARS-CoV-2 nsp8 in Human Lung (SARS-CoV-2 RNA+) tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with formaldehyde, antigen retrieval was by heat mediation in a EDTA Buffer (pH 9). Samples were incubated with primary antibody (1/100) for 45 minutes at 24°C. A Rabbit HRP-conjugated secondary antibody was used.

This image is courtesy of an Abreview submitted by Dr, Aron Emmi

• ELISA

Lab

ELISA - Anti-SARS-CoV2 orf8 antibody [EPR24851-65] - BSA and Azide free (AB284042)

This data was developed using ab283914, the same antibody clone in a different buffer formulation.

Indirect ELISA showing primary antibody ab283914 binding to SARS-CoV2 ORF8 (AA 16-121) protein (His tag). Plates were coated with SARS-CoV2 ORF8 (AA 16-121) protein (His tag) and Recombinant SARS-CoV2 ORF7a Fc Chimera protein at 1000 ng/ml. Binding of ab283914 was assessed in a serial dilution range 0.016- 1000 ng/mL (a 3-fold serial dilution).

Binding was detected using pre-adsorbed secondary antibody, goat anti-rabbit IgG H&L (HRP, ab97080) at 1/2000 dilution.

• WB

Lab

Western blot - Anti-SARS-CoV2 orf8 antibody [EPR24851-65] - BSA and Azide free (AB284042)

This data was developed using ab283914, the same antibody clone in a different buffer formulation.

Lanes 1 & 2 : Red – loading control ab18184 (Mouse monoclonal [HIS.H8] to 6X His tag®) observed at 27 kDa.

Lanes 3 & 4 : Green – ab283914 observed at 27 kDa.

ab283914 was shown to bind specifically to SARS-CoV-2 ORF8 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 (ab18184) 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).

Lanes 1 - 2:

Western blot - Anti-SARS-CoV2 orf8 antibody [EPR24851-65] - BSA and Azide free (ab284042)

Lanes 3 - 4:

Western blot - Anti-SARS-CoV2 orf8 antibody [EPR24851-65] - BSA and Azide free (ab284042) at 1/20000 dilution

Lanes 1 and 3:

SARS-CoV-2 ORF8 (AA 16-121) protein (His tagged) at 0.5 µg

Lanes 2 and 4:

SARS-CoV-2 ORF8 (AA 16-121) protein (His tagged) at 0.2 µg

false