Anti-Met (c-Met) antibody [SP44] - C-terminal

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-Met (c-Met) antibody [SP44] - C-terminal (AB227637)

Flow cytometry overlay histogram showing wild-type HAP1 (green line) and MET knockout HAP1 cells stained with ab227637 (red line). The cells were fixed with 4% formaldehyde (10 min) and then permeabilized with 0.1% PBS-Triton X-100 for 15 min. The cells were then incubated in 1x PBS containing 10% normal goat serum to block non-specific protein-protein interaction followed by the antibody (ab227637) (1x10⁶ in 100 μl at 0.04 μg/ml) for 30 min at 22°C.

The secondary antibody Goat anti-rabbit IgG H&L (Alexa Fluor^® 488, pre-adsorbed) (ab150081) was used at 1/2000 for 30 min at 22°C.

Isotype control antibody was Rabbit IgG (monoclonal) (ab172730) used at the same concentration and conditions as the primary antibody (wild-type HAP1 - black line MET knockout HAP1 - grey line). Unlabelled sample was also used as a control (this line is not shown for the purpose of simplicity).

Acquisition of >5000 events were collected using a .

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Met (c-Met) antibody [SP44] - C-terminal (AB227637)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human colon carcinoma tissue sections labeling Met (c-Met) with ab227637 at 1/50 dilution (4.52 µg/ml). Heat mediated antigen retrieval was performed Heat mediated antigen retrieval with sodium citrate buffer (pH 6.0, epitope retrieval solution 1) for 10 mins. Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) was used as the secondary antibody. Negative control : PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-Met (c-Met) antibody [SP44] - C-terminal (AB227637)

Flow Cytometry analysis of HepG2 (Human liver hepatocellular carcinoma cell line) cells labeling Met (c-Met) with purified ab227637 at 1/20 dilution (11.3 μg/ml) (red). Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) secondary antibody was used at 1/2000 dilution. Isotype control - Rabbit monoclonal IgG (ab172730) / Black. Unlabeled control - Unlabelled cells / blue.

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-Met (c-Met) antibody [SP44] - C-terminal (AB227637)

Flow cytometric analysis of HT-29 (Human colorectal adenocarcinoma cell line) cell line labeling Met (c-Met) with ab227637 at 1/100 dilution (green) compared with a negative control of rabbit IgG (blue).

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Met (c-Met) antibody [SP44] - C-terminal (AB227637)

Formalin-fixed, paraffin-embedded human colon adenocarcinoma tissue stained for Met (c-Met) using ab227637 at 1/50 dilution in immunohistochemical analysis.

• WB

Lab

Western blot - Anti-Met (c-Met) antibody [SP44] - C-terminal (AB227637)

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

c-Met can undergo glycosylation, resulting in a higher molecular weight. It can also be cleaved into α and β chains (PMID : 34210960). The β chain undergoes degradation, resulting in lower molecular weight fragments (PMID : 32522525). This antibody detects both the full-length c-Met and the β chains.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/1000000 dilution.

All lanes:

Western blot - Anti-Met (c-Met) antibody [SP44] - C-terminal (ab227637) at 1/1000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

MET knockout HeLa cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Observed band size: 145-175 kDa,35-55 kDa

false

Exposure time: 60s

• WB

Lab

Western blot - Anti-Met (c-Met) antibody [SP44] - C-terminal (AB227637)

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

The β chain undergoes degradation, resulting in lower molecular weight fragments (PMID : 32522525). To minimize protein degradation, cells/tissues were lysed immediately after harvest and then applied to a gel and transfer membrane for Western blotting right away.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/1000000 dilution.

All lanes:

Western blot - Anti-Met (c-Met) antibody [SP44] - C-terminal (ab227637) at 1/1000 dilution

Lane 1:

Fresh HeLa cell lysate at 20 µg

Lane 2:

Frozen HeLa cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Observed band size: 145-175 kDa,35-55 kDa

false

Exposure time: 60s