Anti-Met (c-Met) antibody [EPR19554-110]

• WB

Lab

Western blot - Anti-Met (c-Met) antibody [EPR19554-110] (AB254252)

Anti-MET antibody [EPR19554-110] (ab254252) staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab254252 was shown to bind specifically to MET. A band was observed at 80-140 kDa in wild-type HeLa cell lysates with no signal observed at this size in MET knockout cell line. 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 Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution. The full length MET protein is glycosylated and has multiple cleavage sites, so cleaved products can be detected between ~40-140 kDa

All lanes:

Western blot - Anti-Met (c-Met) antibody [EPR19554-110] (ab254252) at 1/1000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

MET (Met (c-Met)) knockout HeLa cell lysate at 20 µg

Lane 3:

HT-29 cell lysate at 20 µg

Lane 4:

T-47D cell lysate at 20 µg

Secondary

All lanes:

Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

Observed band size: 80-140 kDa

false

• WB

Lab

Western blot - Anti-Met (c-Met) antibody [EPR19554-110] (AB254252)

The molecular weight observed is consistent with what has been described in the literature (PMID 8710887).

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

All lanes:

Western blot - Anti-Met (c-Met) antibody [EPR19554-110] (ab254252) at 1/1000 dilution

All lanes:

B16-F10 (mouse melanoma mixture of spindle-shaped and epithelial-like cells) whole cell lysate at 10 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 1/100000 dilution

Predicted band size: 155 kDa

Observed band size: 175 kDa

false

• WB

Lab

Western blot - Anti-Met (c-Met) antibody [EPR19554-110] (AB254252)

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

c-Met is an N-linked glycosylated protein. After partial de-glycosylation, the sizes of the pro c-Met (175kDa) nd P125 c-Met (125kDa) are shifted to 160, 140kDa and 80kDa (PMID : 23060267, PMID : 18187039).

All lanes:

Western blot - Anti-Met (c-Met) antibody [EPR19554-110] (ab254252) at 1/1000 dilution

Lane 1:

Untreated B16-F10 (mouse melanoma mixture of spindle-shaped and epithelial-like cells) whole cell lysate at 10 µg

Lane 2:

B16-F10 whole cell lysate treated with PNGase F at 10 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 1/100000 dilution

Predicted band size: 155 kDa

Observed band size: 175 kDa,175-125 kDa

false

Exposure time: 37s