Anti-Notch1 antibody [EP1238Y]

• WB

Lab

Western blot - Anti-Notch1 antibody [EP1238Y] (AB52627)

Western blot : Anti-NOTCH1 antibody [EP1238Y] (ab52627) staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab52627 was shown to bind specifically to NOTCH1. A band was observed at 110-120 kDa in wild-type HL-60 cell lysates with no signal observed at this size in NOTCH1 knockout cell line. To generate this image, wild-type and NOTCH1 knockout HL-60 cell lysates were analysed. First, 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 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

All lanes:

Western blot - Anti-Notch1 antibody [EP1238Y] (ab52627) at 1/1000 dilution

Lane 1:

Wild-type HL-60 cell lysate at 20 µg

Lane 2:

NOTCH1 knockout HL-60 cell lysate at 20 µg

Lane 3:

Wild-type HAP1 cell lysate at 20 µg

Lane 4:

NOTCH1 knockout HAP1 cell lysate at 20 µg

Lane 5:

MOLT-4 cell lysate at 20 µg

Lane 6:

HepG2 cell lysate at 20 µg

Observed band size: 110-120 kDa

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• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-Notch1 antibody [EP1238Y] (AB52627)

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.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Notch1 antibody [EP1238Y] (AB52627)

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-rabbit was used at a dilution of 1/500 as the negative control and is shown in the bottom right hand panel.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Notch1 antibody [EP1238Y] (AB52627)

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.

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

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-Notch1 antibody [EP1238Y] (AB52627)

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-rabbit was used at a dilution of 1/500 as the negative control and is shown in the bottom right hand panel.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Notch1 antibody [EP1238Y] (AB52627)

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.

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

• WB

Lab

Western blot - Anti-Notch1 antibody [EP1238Y] (AB52627)

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

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

All lanes:

Western blot - Anti-Notch1 antibody [EP1238Y] (ab52627) at 1/1000 dilution

Lane 1:

HeLa (human cervical adenocarcinoma epithelial cell)

Lane 2:

293T (human embryonic kidney epithelial cell)

Lane 3:

NIH/3T3 (mouse embryonic fibroblast)

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: 110 kDa,272 kDa,36 kDa

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Exposure time: 180s

• WB

Lab

Western blot - Anti-Notch1 antibody [EP1238Y] (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] (ab52627) 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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• WB

Lab

Western blot - Anti-Notch1 antibody [EP1238Y] (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 3% 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] (ab52627) 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] (AB52627)

Lanes 1- 2 : Merged signal (red and green). Green - ab52627 observed at 110-120 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

ab52627 was shown to react with Notch1 in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line ab261762 (knockout cell lysate ab257006) was used. Wild-type HeLa and NOTCH1 knockout HeLa cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab52627 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) overnight at 4°C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^®800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^®680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-Notch1 antibody [EP1238Y] (ab52627) at 1/1000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

Western blot - Human NOTCH1 knockout HeLa cell line (<a href='/en-us/products/cell-lines/human-notch1-knockout-hela-cell-line-ab261762'>ab261762</a>)

Lane 2:

NOTCH1 knockout HeLa 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] (AB52627)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-Notch1 antibody [EP1238Y] (ab52627) at 1/2000 dilution

All lanes:

HEK-293 (Human epithelial cell line from embryonic kidney) cell lysate at 10 µg

Secondary

All lanes:

HRP goat anti-rabbit (H+L) at 1/1000 dilution

Predicted band size: 272 kDa

Observed band size: 110-120 kDa

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• ChIC/CUT&RUN-seq

Supplier Data

ChIC/CUT&RUN sequencing - Anti-Notch1 antibody [EP1238Y] (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.

• ChIC/CUT&RUN-seq

Supplier Data

ChIC/CUT&RUN sequencing - Anti-Notch1 antibody [EP1238Y] (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).

• WB

Lab

Western blot - Anti-Notch1 antibody [EP1238Y] (AB52627)

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

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

Low expression : Ovary.

All lanes:

Western blot - Anti-Notch1 antibody [EP1238Y] (ab52627) at 1/1000 dilution

Lane 1:

Mouse brain tissue lysate at 40 µg

Lane 2:

Mouse heart tissue lysate at 40 µg

Lane 3:

Mouse spleen tissue lysate at 40 µg

Lane 4:

Mouse lung tissue lysate at 40 µg

Lane 5:

Mouse ovary tissue lysate at 40 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: 110 kDa,36 kDa

false

Exposure time: 180s

• WB

Supplier Data

Western blot - Anti-Notch1 antibody [EP1238Y] (AB52627)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-Notch1 antibody [EP1238Y] (ab52627) at 1/2000 dilution

All lanes:

Mouse brain at 10 µg

Secondary

All lanes:

HRP goat anti-rabbit (H+L) at 1/1000 dilution

Predicted band size: 272 kDa

Observed band size: 110-120 kDa

false

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Notch1 antibody [EP1238Y] (AB52627)

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.