Anti-Notch1 antibody [EP1238Y] - Low endotoxin, Azide free

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-Notch1 antibody [EP1238Y] - Low endotoxin, Azide free (AB246693)

Intracellular Flow Cytometry analysis of permeabilized HeLa (Human epithelial cell line from cervix adenocarcinoma) cells (2% PFA, pink) withpurified ab52627 at a 1/200 dilution, or negative control rabbit monoclonal IgG (green). The secondary antibody was FITC goat anti-rabbit.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab52627).

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Notch1 antibody [EP1238Y] - Low endotoxin, Azide free (AB246693)

Immunohistochemical staining of paraffin-embedded human brain with purified ab52627 at a dilution of 1/150.

A prediluted HRP polymer for rabbit IgG was used as the secondary and the sample was stained with hematoxylin. PBS was used instead of the primary antibody as the negative control, and is shown in the inset.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab52627).

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Notch1 antibody [EP1238Y] - Low endotoxin, Azide free (AB246693)

Immunohistochemical staining of paraffin-embedded human brain with unpurified ab52627 at a dilution of 1/100.

A prediluted HRP polymer for rabbit IgG was used as the secondary and the sample was stained with hematoxylin. PBS was used instead of the primary antibody as the negative control, and is shown in the inset.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab52627).

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Notch1 antibody [EP1238Y] - Low endotoxin, Azide free (AB246693)

Immunofluorescent staining of HeLa (Human epithelial cell line from cervix adenocarcinoma) cells fixed with 4% PFA using unpurified ab52627 at a dilution of 1/100.

An Alexa Fluor^® 555 goat anti-rabbit was used as the secondary and the sample was stained with DAPI. An Alexa Fluor^® 555 goat anti-mouse was used at a dilution of 1/500 as the negative control and is shown in the bottom right hand panel.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab52627).

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-Notch1 antibody [EP1238Y] - Low endotoxin, Azide free (AB246693)

Immunofluorescent staining of HeLa (Human epithelial cell line from cervix adenocarcinoma) cells fixed with 4% PFA using purified ab52627 at a dilution of 1/150.

An Alexa Fluor^® 555 goat anti-rabbit was used as the secondary and the sample was stained with DAPI. An Alexa Fluor^® 555 goat anti-mouse was used at a dilution of 1/500 after ab52627 as the negative control and is shown in the bottom right hand panel.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab52627).

• WB

Lab

Western blot - Anti-Notch1 antibody [EP1238Y] - Low endotoxin, Azide free (AB246693)

This data was developed using the same antibody clone in a different buffer formulation (ab52627).

Lanes 1 - 4 : Merged signal (red and green). Green - ab52627 observed at 110-120 kDa. Red - loading control, ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37kDa.

ab52627 was shown to react with Notch1 in wild-type HAP1 cells in western blot. Loss of signal was observed when NOTCH1 knockout sample was used. Wild-type and NOTCH1 knockout HAP1 cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3pc milk before incubation with ab52627 and ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4°C at 1 μg/ml and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-Notch1 antibody [EP1238Y] (<a href='/en-us/products/primary-antibodies/notch1-antibody-ep1238y-ab52627'>ab52627</a>) at 1 µg/mL

Lane 1:

Wild-type HAP1 cell lysate at 40 µg

Lane 2:

NOTCH1 knockout HAP1 cell lysate at 40 µg

Lane 3:

MOLT-4 cell lysate at 20 µg

Lane 4:

HepG2 cell lysate at 20 µg

Predicted band size: 272 kDa

Observed band size: 110-120 kDa

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• WB

Lab

Western blot - Anti-Notch1 antibody [EP1238Y] - Low endotoxin, Azide free (AB246693)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab52627).

Western blot : Rabbit Monoclonal [EP1238Y] to Notch1 ab52627 staining at 1/1000 dilution, shown in green; Mouse anti alpha Tubulin ab7291 loading control staining at 1/20000 dilution, shown in magenta. A band was observed at 105 kDa in Wild-type HCT 116 cell lysates with no signal observed at this size in NOTCH1 knockout HCT 116 cell line. To generate this image, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 5pc 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 800CW and Goat anti-Mouse 680RD at 1/20,000 dilution.

All lanes:

Western blot - Anti-Notch1 antibody [EP1238Y] (<a href='/en-us/products/primary-antibodies/notch1-antibody-ep1238y-ab52627'>ab52627</a>) at 1/1000 dilution

Lane 1:

Wild-type HCT 116 whole cell lysate at 20 µg

Lane 2:

Western blot - Human NOTCH1 knockout HCT116 cell line (<a href='/en-us/products/cell-lines/human-notch1-knockout-hct116-cell-line-ab287652'>ab287652</a>) at 20 µg

Lane 3:

MOLT-4 whole cell lysate at 20 µg

Lane 4:

HepG2 whole cell lysate at 20 µg

Secondary

All lanes:

Goat anti-Rabbit 800CW & Goat anti-Mouse 680RD at 1/20000 dilution

Predicted band size: 100 kDa

Observed band size: 105 kDa

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• ChIC/CUT&RUN-seq

Supplier Data

ChIC/CUT&RUN sequencing - Anti-Notch1 antibody [EP1238Y] - Low endotoxin, Azide free (AB246693)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab52627).

CUT&RUN profiling with Notch1 antibody reveals the expected genomic enrichment pattern in wild-type (WT) cells, which is substantially reduced in NOTCH1 knockout (KO) cells. Representative genome browser tracks show CUT&RUN data generated using the CUTANA™ CUT&RUN Kit (EpiCypher 14-1048) with Notch1 antibody (Abcam ab52627, 0.5 µg). 500,000 HeLa WT or KO (ab261762) cells were used per reaction. IgG, H3K4me3, and H3K27me3 antibodies were included as controls to assess non-specific background, active promoters, and repressed chromatin, respectively. Libraries were prepared using the CUTANA™ CUT&RUN Library Prep Kit (EpiCypher 14-1001). Sequencing was performed with paired-end 50 bp reads, and data were processed on CUTANA™ Cloud (cloud.epicypher.com) by alignment to the hg38 genome. Images were generated using Integrative Genomics Viewer (IGV, Broad Institute).

• ChIC/CUT&RUN-seq

Supplier Data

ChIC/CUT&RUN sequencing - Anti-Notch1 antibody [EP1238Y] - Low endotoxin, Azide free (AB246693)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab52627).

CUT&RUN profiling with Notch1 antibody demonstrates robust genome-wide enrichment in wild-type (WT) cells, which is markedly diminished in NOTCH1 knockout (KO) cells. Heatmaps of genome-wide signal flanking annotated transcription start sites (TSSs, +/- 2 kbp) display CUT&RUN data generated using the CUTANA™ CUT&RUN Kit (EpiCypher 14-1048) with Notch1 antibody (Abcam ab52627, 0.5 µg). 500,000 HeLa WT or KO (Abcam ab261762) cells were used per reaction. IgG antibody was included as a negative control to assess non-specific background. Libraries were prepared using the CUTANA™ CUT&RUN Library Prep Kit (EpiCypher 14-1001). Sequencing was performed with paired-end 50 bp reads, and data were processed on CUTANA™ Cloud (cloud.epicypher.com) by alignment to the hg38 genome. Heatmaps were generated using deepTools (Ramнrez et al., Nucleic Acids Res. 2014; PMID : 24799436). Row-linked data are ranked by intensity relative to Notch1 WT, with red indicating high localized enrichment and blue denoting background. Validated antibodies show genome-wide enrichment above IgG background consistent with Notch1 binding in WT cells and near complete loss of signal in KO cells.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Notch1 antibody [EP1238Y] - Low endotoxin, Azide free (AB246693)

Verification of gene expression array data by immunohistochemical analysis of Notch 1 expression in subcutaneous tumors and lung metastases from a human melanoma (MeWo) xenograft experiment in mice.

Immunohistochemical staining for Notch1 expression (ab52627, red) in subcutaneous tumors and lung metastases (both panels) of MeWo (Human malignant melanoma cell line) cells.

All scale bars : 50 μm.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab52627).

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.