All lanes : Anti-Notch1 antibody [EP1238Y] (ab52627) at 1/1000 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : NOTCH1 knockout HeLa cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Lanes 1- 2: Merged signal (red and green). Green - ab52627 observed at 110 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.

Decrease of goblet cells in mice fed a high-fat diet (HFD).

Immunohistochemistry using anti-Notch intracellular domain (NICD) ab52627 and phospho-S6 Abs.

Mice intestines were flushed with phosphate-buffered saline (PBS) and fixed in 10% neural formalin overnight at room temperature. The paraffin-embedded specimens were cut into 5 μm sections and stained with hematoxylin and eosin (H&E) or periodic acid-Schiff (PAS)/Alcian blue. Paneth cells were stained with purple, and goblet cells blue with the PAS/Alcian blue method.

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.

All lanes : 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

Performed under reducing conditions.

Observed band size: 105 kDa why is the actual band size different from the predicted?

Lanes 1 - 4: Merged signal (red and green). Green - ab52627 observed at 105 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.

Anti-Notch1 antibody [EP1238Y] (ab52627) at 1/2000 dilution (Purified) + Mouse brain at 10 µg

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

Observed band size: 125 kDa why is the actual band size different from the predicted?

Blocking/Dilution buffer: 5% NFDM/TBST.

 

Anti-Notch1 antibody [EP1238Y] (ab52627) at 1/2000 dilution (Purified) + HEK-293 (Human epithelial cell line from embryonic kidney) cell lysate at 10 µg

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

Observed band size: 125 kDa why is the actual band size different from the predicted?

Blocking/Dilution buffer: 5% NFDM/TBST.

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.

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.

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.

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.

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