Recombinant Anti-Met (c-Met) antibody [EP1454Y] - N-terminal (ab51067)

Lane 1: Wild-type HAP1 cell lysate (40 µg)
Lane 2: Met (c-Met) knockout HAP1 cell lysate (40 µg)
Lane 3: HepG2 cell lysate (40µg) (40 µg)
Lane 4: HEK-293 cel lysate (40µg) (40 µg)

Lanes 1 - 4: Merged signal (red and green). Green - ab51067 observed at 240 kDa. Red - loading control, ab18058, observed at 124 kDa.

This western blot image is a comparison between ab51067 and a competitor's top cited rabbit polyclonal antibody.

ab51067 staining Met (c-Met) in human breast tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections).

Tissue was fixed with formaldehyde, permeabilized with 0.05% Tween-20 and blocked for 30 minutes at 22°C; antigen retrieval was by heat mediation in antigen retrieval buffer (100X citrate buffer pH 6.0) (ab94674). Samples were incubated with the primary antibody (1/100) for 14 hours at 4°C. An Alexa Fluor^® 488-conjugated goat anti-rabbit IgG polyclonal (1/300) was used as the secondary antibody.

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Immunohistochemical staining of paraffin embedded human bladder carcinoma with purified ab51067 at a working dilution of 1/100. The secondary antibody used is HRP goat anti-rabbit IgG H&L (ab97051) at 1/500. The sample is counterstained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0.

PBS was used instead of the primary antibody as the negative control (inset).

Immunohistochemical staining of paraffin embedded human clear cell kidney carcinoma with purified ab51067 at a working dilution of 1/100. The secondary antibody used is HRP goat anti-rabbit IgG H&L (ab97051) at 1/500. The sample is counterstained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0.

PBS was used instead of the primary antibody as the negative control (inset).

Lanes 1 - 3: Merged signal (red and green). Green - ab51067 observed at 240 kDa. Red - loading control, ab18058, observed at 130 kDa.

This blot was produced using a 4-12% Bis-tris gel under the MOPS buffer system. The gel was run at 200V for 50 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using Licor blocking buffer before being incubated with ab51067 and ab18058 (loading control) overnight at 4°C. Antibody binding was detected using Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (ab216776) at a 1:10000 dilution for 1hr at room temperature and then imaged.

Blocking/Dilution buffer: 5% NFDM/TBST.

Immunofluorescence staining of Jurkat (Human T cell leukemia cell line from peripheral blood) cells with purified ab51067 at a working dilution of 1/100, counterstained with DAPI. The secondary antibody was Alexa Fluor^® 488 goat anti-rabbit (ab150077), used at a dilution of 1/1000. ab7291, a mouse anti-tubulin antibody (1/1000), was used to stain tubulin along with ab150120 (Alexa Fluor^® 594 goat anti-mouse, 1/1000), shown in the top right hand panel.

The cells were fixed in 100% methanol and permeabilized using 0.1% Triton X 100.

The negative controls are shown in bottom middle and right hand panels - for negative control 1, purified ab51067 was used at a dilution of 1/500 followed by an Alexa Fluor^® 594 goat anti-mouse antibody (ab150120) at a dilution of 1/500. For negative control 2, ab7291 (mouse anti-tubulin) was used at a dilution of 1/500 followed by an Alexa Fluor^® 488 goat anti-rabbit antibody (ab150077) at a dilution of 1/400.

Overlay histogram showing HeLa (Human epithelial cell line from cervix adenocarcinoma) cells fixed in 4% PFA and stained with purified ab51067 at a dilution of 1 in 100 (red line). The secondary antibody used was FITC goat anti-rabbit at a dilution of 1 in 500. Rabbit monoclonal IgG was used as an isotype control (black line) and cells incubated in the absence of both primary and secondary antibody were used as a negative control (blue line).

Overlay histogram showing Jurkat (Human T cell leukemia cell line from peripheral blood) cells stained with unpurified ab51067 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab51067, 1/1000 dilution) for 30 min at 22°C. The secondary antibody used was a goat anti-rabbit Alexa Fluor^® 488 (IgG, H+L) (ab150077) at 1/2000 dilution for 30 min at 22°C.

Isotype control antibody (black line) was rabbit IgG (monoclonal) (1μg/1x10⁶ cells) used under the same conditions. Unlabeled sample (blue line) was also used as a control.

Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter.

Lane 1: Wild-type HAP1 cell lysate (40 µg)
Lane 2: Met (c-Met) knockout HAP1 cell lysate (40 µg)
Lane 3: HepG2 cell lysate (40µg) (40 µg)
Lane 4: HEK-293 cel lysate (40µg) (40 µg)

Lanes 1 - 4: Merged signal (red and green). Green - ab51067 observed at 240 kDa. Red - loading control, ab18058, observed at 124 kDa.

ab51067 was shown to specifically recognize Met (c-Met) in wild-type HAP1 cells along with additional cross reactive bands. No bands were observed when Met (c-Met) knockout samples were used. Wild-type and Met (c-Met) knockout samples were subjected to SDS-PAGE. ab51067 and ab18058 (loading control to Vinculin) were diluted at 1/1000 and 1/10,000 dilution respectively and incubated overnight at 4C. 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 1/10,000 dilution for 1hr at room temperature before imaging.