Anti-EpCAM antibody [E144]

• WB

Lab

Western blot - Anti-EpCAM antibody [E144] (AB32392)

Lanes 1 - 4 : Merged signal (red and green). Green - ab32392 observed at 40 kDa. Red - loading control ab7291 (Mouse anti-Alpha Tubulin [DM1A] observed at 55kDa.

ab32392 was shown to react with EpCAM in wild-type A431 cells in western blot with loss of signal observed in EpCAM knockout sample. Wild-type and EpCAM knockout A431 cell lysates were subjected to SDS-PAGE. Membranes were blocked in fluorescent western blot (TBS-based) blocking solution before incubation with ab32392 and ab7291 (Mouse anti-Alpha Tubulin [DM1A] overnight at 4°C at a 1 in 2000 dilution and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-EpCAM antibody [E144] (ab32392) at 1/2000 dilution

Lane 1:

Wild-type A431 cell lysate at 20 µg

Lane 2:

EPCAM knockout A431 cell lysate at 20 µg

Lane 2:

Western blot - Human EPCAM knockout A-431 cell line (<a href='/en-us/products/cell-lines/human-epcam-knockout-a-431-cell-line-ab261902'>ab261902</a>)

Lane 3:

MCF7 (Human breast adenocarcinoma cell line) whole cell lysate at 20 µg

Lane 4:

HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 20 µg

Predicted band size: 35 kDa

Observed band size: 40 kDa

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• WB

Lab

Western blot - Anti-EpCAM antibody [E144] (AB32392)

False colour image of Western blot : Anti-EpCAM antibody [E144] staining at 1/1000 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab32392 was shown to bind specifically to EpCAM. A band was observed at 37/45 kDa in wild-type HCT 116 cell lysates with no signal observed at this size in EPCAM knockout cell line ab281596 (knockout cell lysate ab282948). To generate this image, wild-type and EPCAM knockout HCT 116 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 5 % 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 (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) at 1/20000 dilution.

All lanes:

Western blot - Anti-EpCAM antibody [E144] (ab32392) at 1/1000 dilution

Lane 1:

Wild-type HCT 116 cell lysate at 20 µg

Lane 2:

EPCAM knockout HCT 116 cell lysate at 20 µg

Lane 3:

A431 cell lysate at 20 µg

Lane 4:

MCF7 cell lysate at 20 µg

Lane 5:

HeLa cell lysate at 20 µg

Predicted band size: 35 kDa

Observed band size: 37-45 kDa

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• WB

Unknown

Western blot - Anti-EpCAM antibody [E144] (AB32392)

All lanes:

Western blot - Anti-EpCAM antibody [E144] (ab32392) at 1/2500 dilution

All lanes:

A431 cell lysate

Predicted band size: 35 kDa

false

• WB

Unknown

Western blot - Anti-EpCAM antibody [E144] (AB32392)

All lanes:

Western blot - Anti-EpCAM antibody [E144] (ab32392) at 1/1000 dilution

Lane 1:

Mouse colon tissue lysate at 20 µg

Lane 2:

Rat small intestine tissue lysate at 20 µg

Lane 3:

Rat colon tissue 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 1/100000 dilution

Predicted band size: 35 kDa

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