All lanes : Anti-NOTCH4 antibody [EPR18049] (ab184742) at 1/2000 dilution

Lane 1 : HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate
Lane 2 : HepG2 (Human liver hepatocellular carcinoma cell line) whole cell lysate
Lane 3 : A549 (Human lung carcinoma cell line) whole cell lysate
Lane 4 : Jurkat (Human T cell leukemia cell line from peripheral blood) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (Agarose) (ab97052) at 1/100000 dilution

Predicted band size: 210 kDa
Observed band size: 150 kDa why is the actual band size different from the predicted?

Blocking/Dilution buffer: 5% NFDM/TBST.

Exposure time: Lane 1,2 and 3: 15 seconds;Lane 4: 3 minute.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (Human epithelial cell line from cervix adenocarcinoma) cells labeling NOTCH4 with ab184742 at 1/200 dilution, followed by Goat Anti-Rabbit IgG (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green).

Confocal image showing cytoplasmic staining on HeLa cell line.

The nuclear counterstain is DAPI (blue).

Tubulin is detected with ab195889 (Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594)) at 1/200 dilution (red).

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is ab150077 at 1/1000 dilution.

Flow cytometric analysis of 2% paraformaldehyde-fixed HeLa (Human epithelial cell line from cervix adenocarcinoma) cells labeling NOTCH4 with ab184742 at 1/200 dilution (red) compared with a Rabbit IgG,monoclonal [EPR25A]-Isotype Control (ab172730) (black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (blue). Goat anti Rabbit IgG (FITC) at 1/150 dilution was used as the secondary antibody.

All lanes : Anti-NOTCH4 antibody [EPR18049] (ab184742) at 1/2000 dilution

Lane 1 : Human fetal lung lysate
Lane 2 : Human placenta lysate
Lane 3 : Human fetal brain lysate
Lane 4 : Human fetal heart lysate
Lane 5 : Human fetal kidney lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG Peroxidase Conjugate, specific to the non-reduced form of IgG at 1/10000 dilution

Predicted band size: 210 kDa
Observed band size: 150 kDa why is the actual band size different from the predicted?

Blocking/Dilution buffer: 5% NFDM/TBST.

Exposure time: Lane 1,3,4 and 5: 3 minutes; Lane 2: 30 seconds.

The bands beneath the 150 kD NECD band are likely to be the degradation fragments.

Anti-NOTCH4 antibody [EPR18049] (ab184742) at 1/1000 dilution + Human NOTCH4 fragment recombinant protein at 0.01 µg

Secondary
Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 210 kDa


Exposure time: 1 second

Blocking/Dilution buffer: 5% NFDM/TBST.

All lanes : Anti-NOTCH4 antibody [EPR18049] (ab184742) at 1/2000 dilution

Lane 1 : Mouse brain lysate
Lane 2 : Mouse heart lysate
Lane 3 : Mouse spleen lysate
Lane 4 : Rat brain lysate
Lane 5 : Rat heart lysate
Lane 6 : C6 (Rat glial tumor cell line) whole cell lysate
Lane 7 : RAW 264.7 (Mouse macrophage cell line transformed with Abelson murine leukemia virus) whole cell lysate
Lane 8 : PC-12 (Rat adrenal gland pheochromocytoma cell line) whole cell lysate
Lane 9 : NIH/3T3 (Mouse embryonic fibroblast cell line) whole cell lysate
Lane 10 : Mouse placenta lysate
Lane 11 : F9 (Mouse embryonic testicular cancer cell line) whole cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 210 kDa
Observed band size: 150 kDa why is the actual band size different from the predicted?

Blocking/Dilution buffer: 5% NFDM/TBST.

Exposure time: Lane 1,2,3,4 and 5: 1 minute; Lane 6, 7, 8 and 9: 15 seconds; Lane 10 and 11: 30 seconds.

The bands beneath the 150 kD NECD band are likely to be the degradation fragments.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized NIH/3T3 (Mouse embryonic fibroblast cell line) cells labeling NOTCH4 with ab184742 at 1/200 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green).

Confocal image showing cytoplasmic staining on NIH/3T3 cell line.

The nuclear counterstain is DAPI (blue).

Tubulin is detected with ab195889 (Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594)) at 1/200 dilution (red).

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat anti-rabbit IgG (Alexa Fluor^® 488) (ab150077) at 1/1000 dilution.

Flow cytometric analysis of 2% paraformaldehyde-fixed NIH/3T3 (Mouse embryonic fibroblast cell line) cells labeling NOTCH4 with ab184742 at 1/200 dilution (red) compared with a Rabbit IgG,monoclonal [EPR25A]-Isotype control (ab172730) (black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (blue). Goat anti Rabbit IgG (FITC) at 1/150 dilution was used as the secondary antibody.

NOTCH4 was immunoprecipitated from 1mg of HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate with ab184742 at 1/60 dilution.

Western blot was performed from the immunoprecipitate using ab184742 at 1/5000 dilution.

Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG was used as secondary antibody at 1/1500 dilution.

Lane 1: HeLa whole cell lysate, 10µg (Input).

Lane 2: ab184742 IP in HeLa whole cell lysate.

Lane 3: Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) instead of ab184742 in HeLa whole cell lysate.

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

Exposure time: 1 second.