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

• 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.

• 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 .

• 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

Unknown

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.

• IHC-P

Lab

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

Immunohistochemical analysis of paraffin-embedded HeLa wt cell pellet and HeLa MET KO cell pellet labeling MET (c-MET) with ab227637 at 1/50 dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining in human HeLa wt and negative staining in human HeLa MET KO cells. The section was incubated with ab227637 for 15 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument Counterstained with Hematoxylin. Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Heat mediated antigen retrieval was performed with Citrate buffer (pH 6.0, Epitope Retrieval Solution 1) for 20 mins

• 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='/ko/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.

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='/ko/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

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).

ab227637 detects both the full-length c-Met and the β-chain, including β-chain degradation products between 35-55 kDa, and generally produces cleaner Western blot results than ab216574.

Lanes 1 - 2:

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

Lanes 3 - 4:

Western blot - Anti-Met (c-Met) antibody [EPR19067] (<a href='/ko/products/primary-antibodies/met-c-met-antibody-epr19067-ab216574'>ab216574</a>) at 1/1000 dilution

Lanes 1 and 3:

Wild-type HeLa cell lysate at 20 µg

Lanes 2 and 4:

MET knockout HeLa cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/ko/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