Anti-NOTCH3 antibody [RM1102]

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-NOTCH3 antibody [RM1102] (AB315348)

Immunohistochemical analysis of paraffin-embedded Human breast cancer tissue labeling NOTCH3 with ab315348 at 1/500 (1.046 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on human breast cancer. The section was incubated with ab315348 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.

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-NOTCH3 antibody [RM1102] (AB315348)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized HeLa (human cervical adenocarcinoma epithelial cell) cells labelling NOTCH3 with ab315348 at 1/500 dilution (0.1 ug)/Red compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue).

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

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-NOTCH3 antibody [RM1102] (AB315348)

Immunohistochemical analysis of paraffin-embedded (A) HEK-293T (human embryonic kidney epithelial cell) transfected with a human NOTCH3 expression vector containing a His tag. (B) HEK-293T transfected with empty vector containing a His tag. tissue labeling NOTCH3 with ab315348 at 1/5000 (5.23 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on HEK-293T transfected with a human NOTCH3 expression vector (image A). No staining on HEK-293T transfected with empty vector (image B). The section was incubated with ab315348 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.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-NOTCH3 antibody [RM1102] (AB315348)

Immunohistochemical analysis of paraffin-embedded Human endometrium tissue labeling NOTCH3 with ab315348 at 1/500 (1.046 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on human smooth muscle of endometrium. The section was incubated with ab315348 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.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-NOTCH3 antibody [RM1102] (AB315348)

Immunohistochemical analysis of paraffin-embedded Human colon tissue labeling NOTCH3 with ab315348 at 1/500 (1.046 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on human smooth muscle of colon. The section was incubated with ab315348 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-NOTCH3 antibody [RM1102] (AB315348)

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/1000000 dilution.

Lysates were freshly made and used for Western blotting immediately to minimize protein degradation.

This blot was developed using a high sensitivity ECL substrate. The high-sensitivity ECL substrate used allows for the detection of proteins in the mid-femtogram range.

All lanes:

Western blot - Anti-NOTCH3 antibody [RM1102] (ab315348) at 1/1000 dilution

Lane 1:

MCF7 (human breast adenocarcinoma epithelial cell) whole cell lysate (frozen) at 20 µg

Lane 2:

MCF7 (human breast adenocarcinoma epithelial cell) whole cell lysate (fresh) 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 1/20000 dilution

Observed band size: 90 kDa,270 kDa

true

Exposure time: 180s

• WB

Supplier Data

Western blot - Anti-NOTCH3 antibody [RM1102] (AB315348)

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

The expression profile and molecular weight observed are consistent with what has been described in the literature (PMID : 29109797, 24743243, 25356737).

Lysates were freshly made and used for Western blotting immediately to minimize protein degradation.

Exposure time : Lane 1 : 114 seconds; Lane 2 : 59 seconds

All lanes:

Western blot - Anti-NOTCH3 antibody [RM1102] (ab315348) at 1/1000 dilution

Lane 1:

293T (human embryonic kidney epithelial cell) whole cell lysate at 20 µg

Lane 2:

MCF7 (human breast adenocarcinoma epithelial cell) 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 1/20000 dilution

Observed band size: 90 kDa

true

• WB

Supplier Data

Western blot - Anti-NOTCH3 antibody [RM1102] (AB315348)

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

The expression profile and molecular weight observed are consistent with what has been described in the literature (PMID : 29109797, 24743243, 25356737).

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution.

All lanes:

Western blot - Anti-NOTCH3 antibody [RM1102] (ab315348) at 1/1000 dilution

Lane 1:

HeLa (human cervical adenocarcinoma epithelial cell) transfected with scrambled siRNA control whole cell lysate at 20 µg

Lane 2:

HeLa transfected with siRNA specifically targeting NOTCH3 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 1/20000 dilution

Observed band size: 270 kDa,90 kDa,36 kDa

true

Exposure time: 114s