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

Blocking buffer / Diluent and concentration:

5% NFDM/TBST

False colour image of Western blot: Anti-Met (c-Met) antibody [EP1454Y] - N-terminal staining at 1/1000 dilution, shown in green; Rabbit Anti-GAPDH antibody [EPR16891] (ab181602) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab51067 was shown to bind specifically to the alpha chain of c-Met. A band was observed at 50 kDa in wild-type HeLa cell lysates with no signal observed at this size in MET knockout cell line ab265961 (knockout cell lysate ab256991). To generate this image, wild-type and MET knockout HeLa 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 HRP conjugated Goat anti-Rabbit (H+L) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) at 1/20000 dilution.

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).

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 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.

ELISA analysis of Human Met recombinant protein at 1000 ng/mL with ab51067. An Alkaline Phosphatase-conjugated AffiniPure Goat Anti-Rabbit IgG (H+L) at 1/2500 dilution was used as the secondary antibody.

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.