重组Anti-Daxx抗体[E94]

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Daxx antibody [E94] (AB32140)

ab32140, at a dilution of 1/50, staining Daxx in paraffin embedded human stomach adenocarcinoma tissue by Immunohistochemistry.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

• WB

Lab

Western blot - Anti-Daxx antibody [E94] (AB32140)

Lanes 1- 2 : Merged signal (red and green). Green - ab32140 observed at 100 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

ab32140 was shown to react with Daxx in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line ab265233 (knockout cell lysate ab257408) was used. Wild-type HeLa and DAXX knockout HeLa cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab32140 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) overnight at 4°C at a 1 in 5000 dilution and a 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^®800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^®680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-Daxx antibody [E94] (ab32140) at 1/5000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

DAXX knockout HeLa cell lysate at 20 µg

Lane 2:

Western blot - Human DAXX knockout HeLa cell line (<a href='/products/cell-lines/human-daxx-knockout-hela-cell-line-ab265233'>ab265233</a>)

Predicted band size: 81 kDa

Observed band size: 100 kDa

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• WB

Lab

Western blot - Anti-Daxx antibody [E94] (AB32140)

False colour image of Western blot : Anti-Daxx antibody [E94] staining at 1/5000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab32140 was shown to bind specifically to Daxx. A band was observed at 105 kDa in wild-type A549 cell lysates with no signal observed at this size in DAXX knockout cell line. To generate this image, wild-type and DAXX knockout A549 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.

Lanes 1 - 3:

Western blot - Anti-Daxx antibody [E94] (ab32140) at 1/5000 dilution

Lanes 1 - 3:

Western blot - Anti-Daxx antibody [E94] - BSA and Azide free (<a href='/products/primary-antibodies/daxx-antibody-e94-bsa-and-azide-free-ab239806'>ab239806</a>) at 1/5000 dilution

Lane 1:

Wild-type A549 cell lysate at 20 µg

Lane 2:

Western blot - Human DAXX knockout A549 cell line (<a href='/products/cell-lines/human-daxx-knockout-a549-cell-line-ab287356'>ab287356</a>)

Lane 2:

DAXX knockout A549 cell lysate at 20 µg

Lane 3:

THP-1 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: 105 kDa

false

• WB

Unknown

Western blot - Anti-Daxx antibody [E94] (AB32140)

All lanes:

Western blot - Anti-Daxx antibody [E94] (ab32140) at 1/5000 dilution

All lanes:

Hela cell lysate

Predicted band size: 81 kDa

Observed band size: 100 kDa,48 kDa

false

• WB

Supplier Data

Western blot - Anti-Daxx antibody [E94] (AB32140)

False colour image of Western blot : Anti-Daxx antibody [E94] staining at 1/5000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab32140 was shown to bind specifically to Daxx. A band was observed at 105 kDa in wild-type MCF7 cell lysates with no signal observed at this size in DAXX knockout cell line. To generate this image, wild-type and DAXX knockout MCF7 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 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

All lanes:

Western blot - Anti-Daxx antibody [E94] (ab32140) at 1/5000 dilution

Lane 1:

Wild-type MCF7 cell lysate at 20 µg

Lane 2:

DAXX knockout MCF7 cell lysate at 20 µg

Lane 3:

THP-1 cell lysate at 20 µg

Lane 4:

K562 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

Predicted band size: 81 kDa

Observed band size: 105 kDa

false

• WB

Lab

Western blot - Anti-Daxx antibody [E94] (AB32140)

Lanes 1 - 2 : Merged signal (red and green). Green - ab32140 observed at 100 kDa. Red - loading control, ab9484, observed at 37 kDa.

ab32140 was shown to specifically react with Daxx in wild-type HAP1 cells as signal was lost in DAXX knockout cells. Wild-type and DAXX knockout samples were subjected to SDS-PAGE. ab32140 and ab9484 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 1/5000 dilution and 1/20000 dilution respectively. Blots were developed 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 1/20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-Daxx antibody [E94] (ab32140)

Lane 1:

Wild-type HAP1 whole cell lysate at 20 µg

Lane 2:

DAXX knockout HAP1 whole cell lysate at 20 µg

Predicted band size: 81 kDa

false

• WB

Lab

Western blot - Anti-Daxx antibody [E94] (AB32140)

False colour image of Western blot : Anti-Daxx antibody [E94] staining at 1/5000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab32140 was shown to bind specifically to Daxx. A band was observed at 100 kDa in wild-type HCT 116 cell lysates with no signal observed at this size in DAXX knockout cell line. To generate this image, wild-type and DAXX 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 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-Daxx antibody [E94] (ab32140) at 1/5000 dilution

Lane 1:

Wild-type HCT 116 cell lysate at 20 µg

Lane 2:

Western blot - Human DAXX knockout HCT116 cell line (<a href='/products/cell-lines/human-daxx-knockout-hct116-cell-line-ab287355'>ab287355</a>)

Lane 2:

DAXX knockout HCT 116 cell lysate at 20 µg

Lane 3:

THP-1 cell lysate at 20 µg

Lane 4:

K562 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

Predicted band size: 81 kDa

Observed band size: 100 kDa

false

• WB

Lab

Western blot - Anti-Daxx antibody [E94] (AB32140)

Blocking and diluting buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-Daxx antibody [E94] (ab32140) at 0.271 µg/mL

Lane 1:

PC-12(Rat adrenal gland pheochromocytoma) whole cell lysate at 20 µg

Lane 2:

NIH/3T3(Mouse embryonic fibroblast) whole cell lysate at 20 µg

Secondary

All lanes:

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

Predicted band size: 81 kDa

false

Exposure time: 29s