Anti-Bat coronavirus HKU5 Spike protein S1 (RBD domain) antibody [EPR31043-570]

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-Bat coronavirus HKU5 Spike protein S1 (RBD domain) antibody [EPR31043-570] (AB326881)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized HEK-293T (human epithelial cell line from embryonic kidney) transfected with a Bat coronavirus Spike protein S1 expression vector containing a His-Myc tag® (Middle) and 293T cells transfected with an empty expression vector containing a His-Myc tag® (Right) cells labelling with ab326881 at 1/5000 dilution (0.01ug) / Middle and Right compared with a Rabbit monoclonal IgG (ab172730) / Left isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody).

Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/5000 dilution was used as the secondary antibody.

Cells were co-stained with Myc tag conjugated to Alexa Fluor®647.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Bat coronavirus HKU5 Spike protein S1 (RBD domain) antibody [EPR31043-570] (AB326881)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized HEK-293T cells transfected with an empty expression vector containing a myc-His-tag® and HEK-293T (human epithelial cell line from embryonic kidney) transfected with a Bat coronavirus Spike protein S1 expression vector containing a His-Myc tag cells , labelling with ab326881 at 1/1000 (0.479 µg/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 dilution (Green).

Confocal image showing positive staining in HEK-293T transfected with a Bat coronavirus Spike protein S1 expression vector containing a His-Myc tag(shown in green). The counterstain was observed in magenta. Nuclear DNA was labelled with DAPI (shown in blue). Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

ab223894 Anti-Myc tag mouse monoclonal antibody (Alexa Fluor® 594) was used to counterstain tubulin at 1/100 dilution (Magenta). The Nuclear counterstain was DAPI (Blue).

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Bat coronavirus HKU5 Spike protein S1 (RBD domain) antibody [EPR31043-570] (AB326881)

Immunohistochemical analysis of paraffin-embedded
(A) HEK-293T (human epithelial cell line from embryonic kidney) transfected with a Bat coronavirus Spike protein S1 expression vector containing a His-Myc tag
(B) HEK-293T transfected with empty vector containing a His-Myc tag
labeling with ab326881 at 1/500 (0.958 µg/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive on (A) HEK-293T transfected with a Bat coronavirus Spike protein S1 expression vector containing a His-Myc tag, no staining on (B) HEK-293T transfected with empty vector containing a His-Myc tag. The primary antibody was incubated 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).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• WB

Lab

Western blot - Anti-Bat coronavirus HKU5 Spike protein S1 (RBD domain) antibody [EPR31043-570] (AB326881)

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

This antibody does not cross-react with overexpressed MERS-CoV Spike protein S1 by western blot.

This antibody detects transfected levels of Bat coronavirus Spike protein S1 only and the immunogen used targets the RBD domain.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa); Anti-6X His tag® antibody [EPR20547] - ChIP Grade (ab213204) (1 : 5000)(81KDa).

All lanes:

Western blot - Anti-Bat coronavirus HKU5 Spike protein S1 (RBD domain) antibody [EPR31043-570] (ab326881) at 1/1000 dilution

Lane 1:

293T (human embryonic kidney epithelial cell) cells transfected with an empty vector containing a myc-His-tag® whole cell lysate at 20 µg

Lane 2:

293T cells transfected with a Bat coronavirus Spike protein S1 expression vector containing a myc-His-tag® whole cell lysate at 40 µg

Lane 3:

293T cells transfected with a MERS-CoV Spike protein S1 expression vector containing a myc-His-tag® whole cell lysate at 5 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: 81 kDa,36 kDa

false

Exposure time: 180s