Anti-HE4 antibody [EPR16658]

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-HE4 antibody [EPR16658] (AB200828)

Immunohistochemical analysis of paraffin-embedded Human ovarian carcinoma labeling HE4 with ab200828 at 1/1000 dilution followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Cytoplasmic staining on Human ovarian carcinoma tissue is observed.

Counter stained with Hematoxylin.
Negative control : Used PBS instead of primary ab, secondary ab is Goat Anti-Rabbit IgG H&L (HRP) (ab97051).

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• IP

Supplier Data

Immunoprecipitation - Anti-HE4 antibody [EPR16658] (AB200828)

HE4 was immunoprecipitated from 1mg of NIH : OVCAR-3 (Human ovary adenocarcinoma ) whole cell lysate with ab200828 at 1/120 dilution.

Western blot was performed from the immunoprecipitate using ab200828 at 1/1000 dilution.

Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG, was used as secondary antibody at 1/1500 dilution.

Lane 1 : NIH : OVCAR-3 (Human ovary adenocarcinoma) whole cell lysate 10μg.

Lane 2 : NIH : OVCAR-3 (Human ovary adenocarcinoma) whole cell lysate following IP.

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab200828 in NIH : OVCAR-3 (human ovary adenocarcinoma) whole cell lysate.
Blocking and dilution buffer and concentration : 5% NFDM/TBST. 10 second exposure.

All lanes:

Immunoprecipitation - Anti-HE4 antibody [EPR16658] (ab200828)

Predicted band size: 13 kDa

Observed band size: 17-25 kDa

false

• WB

Supplier Data

Western blot - Anti-HE4 antibody [EPR16658] (AB200828)

Blocking/dilution buffer : 5% NFDM/TBST.

The expression profile observed is consistent with what has been described in the literature PMID : 15781627

All lanes:

Western blot - Anti-HE4 antibody [EPR16658] (ab200828) at 1/1000 dilution

Lane 1:

Human ovary cancer whole cell lysate at 10 µg

Lane 2:

NIH:OVCAR-3 (Human ovary adenocarcinoma) whole cell lysate at 10 µg

Secondary

All lanes:

Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/1000 dilution

Predicted band size: 13 kDa

Observed band size: 17-25 kDa

false

Exposure time: 1min

• WB

Lab

Western blot - Anti-HE4 antibody [EPR16658] (AB200828)

False colour image of Western blot : Anti-HE4 antibody [EPR16658] staining at 1/2000 dilution, shown in black; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab200828 was shown to bind specifically to HE4. 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 before development with Optiblot (ECL reagent ab133456) and imaged with 2 minutes 30 seconds exposure time. 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.

All lanes:

Western blot - Anti-HE4 antibody [EPR16658] (ab200828) at 1/2000 dilution

Lane 1:

OVCAR-3 cell lysate at 20 µg

Lane 2:

K562 cell lysate at 20 µg

Predicted band size: 13 kDa

Observed band size: 20 kDa

false

• WB

Lab

Western blot - Anti-HE4 antibody [EPR16658] (AB200828)

False colour image of Western blot : Anti-HE4 antibody [EPR16658] staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab200828 was shown to bind specifically to HE4. A band was observed at 20 kDa in treated wild-type OVCAR-3 cell lysates with no signal observed at this size in WFDC2 knockout cell line ab275410 (knockout cell lysate ab275536). To generate this image, wild-type and WFDC2 knockout OVCAR-3 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.

All lanes:

Western blot - Anti-HE4 antibody [EPR16658] (ab200828) at 1/1000 dilution

Lane 1:

Wild-type OVCAR-3 Treated Brefeldin A (<a href='/en-us/products/sample-preparation-kits/brefeldin-a-solution-1000x-ab193369'>ab193369</a>) (5 ug/mL, 4 h), Monensin (<a href='/en-us/products/sample-preparation-kits/monensin-solution-1000x-ab193381'>ab193381</a>) (2 uM, 4 h) cell lysate at 20 µg

Lane 2:

Wild-type OVCAR-3 Vehicle Control Brefeldin A (<a href='/en-us/products/sample-preparation-kits/brefeldin-a-solution-1000x-ab193369'>ab193369</a>) (0 ug/mL, 4 h), Monensin (<a href='/en-us/products/sample-preparation-kits/monensin-solution-1000x-ab193381'>ab193381</a>) (0 uM, 4 h) cell lysate at 20 µg

Lane 3:

WFDC2 knockout OVCAR-3 Treated Brefeldin A (<a href='/en-us/products/sample-preparation-kits/brefeldin-a-solution-1000x-ab193369'>ab193369</a>) (5 ug/mL, 4 h), Monensin (<a href='/en-us/products/sample-preparation-kits/monensin-solution-1000x-ab193381'>ab193381</a>) (2 uM, 4 h) cell lysate at 20 µg

Lane 4:

WFDC2 knockout OVCAR-3 Vehicle Control Brefeldin A (<a href='/en-us/products/sample-preparation-kits/brefeldin-a-solution-1000x-ab193369'>ab193369</a>) (0 ug/mL, 4 h), Monensin (<a href='/en-us/products/sample-preparation-kits/monensin-solution-1000x-ab193381'>ab193381</a>) (0 uM, 4 h) cell lysate at 20 µg

Observed band size: 20 kDa

false