All lanes : Anti-Met (c-Met) antibody [EPR19067] (ab216574) at 1/1000 dilution

Lane 1 : Wild-type HAP1 whole cell lysate
Lane 2 : MET knockout HAP1 whole cell lysate

Lysates/proteins at 40 µg per lane.

Predicted band size: 155 kDa

Lanes 1 - 2: Merged signal (red and green). Green - ab216574 observed at 155 kDa. Red - loading control, ab18058, observed at 130 kDa.

ab216574 was shown to specifically recognize MET in wild-type HAP1 cells along with additional cross reactive bands. No bands were observed when MET knockout samples were used. Wild-type and MET knockout samples were subjected to SDS-PAGE. ab216574 and ab18058 (Mouse anti Vinculin loading control) were incubated overnight at 4°C at 1/1000 dilution and 1/10,000 dilution respectively. Blots were developed 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 1/10,000 dilution for 1hr at room temperature before imaging.

Immunohistochemical analysis of paraffin-embedded human breast tissue labeling Met (c-Met) with ab216574 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) ready to use.

Membranous staining on human breast is observed.

Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) ready to use.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (Human epithelial cell line from cervix adenocarcinoma) cells labeling Met (c-Met) with ab216574 at 1/1000 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 Goat anti-rabbit IgG (Alexa Fluor^® 488) (ab150077) at 1/1000 dilution.

All lanes : Anti-Met (c-Met) antibody [EPR19067] (ab216574) at 1/1000 dilution

Lane 1 : A549 (Human lung carcinoma cell line) whole cell lysate
Lane 2 : HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate
Lane 3 : HepG2 (Human liver hepatocellular carcinoma 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: 155 kDa
Observed band size: 45-175 kDa why is the actual band size different from the predicted?


Exposure time: 30 seconds

Blocking/Dilution buffer: 5% NFDM/TBST.

This antibody detects Pro-c-Met (175 kDa), c-Met beta subunit (145 kDa) [PMID: 22418436], c-Met alpha subunit (45 kDa) [PMID: 8710887] and a cleavage c-Met fragment (85 kDa) [PMID: 11786517].

Anti-Met (c-Met) antibody [EPR19067] (ab216574) at 1/1000 dilution + Human liver lysate at 10 µg

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

Predicted band size: 155 kDa
Observed band size: 100-150 kDa why is the actual band size different from the predicted?


Exposure time: 30 seconds

Blocking/Dilution buffer: 5% NFDM/TBST.

In human liver the antibody detected c-Met beta subunit (145 kDa) [PMID: 22418436] and a cleavage c-Met fragment (100 kDa) [PMID: 18187039].

All lanes : Anti-Met (c-Met) antibody [EPR19067] (ab216574) at 1/10000 dilution

Lane 1 : 293T whole cell lysate (Human epithelial cell line from embryonic kidney) transfected with an empty expression vector
Lane 2 : 293T whole cell lysate transfected with a His-tagged human c-Met construct

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: 155 kDa
Observed band size: 150-175 kDa why is the actual band size different from the predicted?


Exposure time: 1 second

Blocking and Diluting buffer and concentration: 5% NFDM /TBST 

All lanes : Anti-Met (c-Met) antibody [EPR19067] (ab216574) at 1/1000 dilution

Lane 1 : Untreated HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate
Lane 2 : HeLa whole cell lysate treated with PNGase F

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: 155 kDa


Exposure time: 1 minute

Blocking/Dilution buffer: 5% NFDM/TBST.

After partial deglycosylation, the band of c-Met beta subunit (145 kDa) is shifted to 125 kDa. The 85 kDa glycosylated band is reduced and a ~60 kDa band appears.

ELISA analysis of Human c-met recombinant protein at 1000 ng/mL with ab216574. An Alkaline Phosphatase-conjugated AffiniPure Goat Anti-Rabbit IgG (H+L) at 1/2500 dilution was used as the secondary antibody.

Immunohistochemical analysis of paraffin-embedded human colon tissue labeling Met (c-Met) with ab216574 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) ready to use.

Membranous staining on human colon is observed.

Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) ready to use.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded human liver cancer tissue labeling Met (c-Met) with ab216574 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) ready to use.

Membranous staining on tumor cells of human liver cancer is observed.

Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) ready to use.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded human ovary cancer tissue labeling Met (c-Met) with ab216574 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) ready to use.

Cytoplasmic and membranous staining on tumor cells of human ovary cancer is observed.

Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) ready to use.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized A549 (Human lung carcinoma cell line) cells labeling Met (c-Met) with ab216574 at 1/1000 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green).

Confocal image showing cytoplasmic staining on A549 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 4% paraformaldehyde-fixed A549 (Human lung carcinoma cell line) cells labeling Met (c-Met) with ab216574 at 1/600 dilution (red) compared with a rabbit monoclonal IgG isotype control (ab172730; black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody; blue). Goat anti rabbit IgG (Alexa Fluor^® 488) at 1/2000 dilution was used as the secondary antibody.

Flow cytometric analysis of 4% paraformaldehyde-fixed HeLa (Human epithelial cell line from cervix adenocarcinoma) cells labeling Met (c-Met) with ab216574 at 1/600 dilution (red) compared with a rabbit monoclonal IgG isotype control (ab172730; black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody; blue). Goat anti rabbit IgG (Alexa Fluor^® 488) at 1/2000 dilution was used as the secondary antibody.