Anti-HDAC4 antibody [EPR22937-157]

• WB

Lab

Western blot - Anti-HDAC4 antibody [EPR22937-157] (AB235583)

Western blot : Anti-HDAC4 antibody [EPR22937-157] (ab235583) 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, ab235583 was shown to bind specifically to HDAC4. A band was observed at 140 kDa in wild-type A549 cell lysates with no signal observed at this size in HDAC4 knockout cell line. To generate this image, wild-type and HDAC4 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.

All lanes:

Western blot - Anti-HDAC4 antibody [EPR22937-157] (ab235583) at 1/1000 dilution

Lane 1:

Wild-type A549 cell lysate at 20 µg

Lane 2:

HDAC4 knockout A549 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: 140 kDa

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• WB

Lab

Western blot - Anti-HDAC4 antibody [EPR22937-157] (AB235583)

ab235583 was shown to specifically react with HDAC4 in wild-type HAP1 cells as signal was lost in HDAC4 knockout cells. Wild-type and HDAC4 knockout samples were subjected to SDS-PAGE.

ab235583 and ab181602 (Rabbit anti-GAPDH loading control) were incubated 1 hour at room temperature at 1/1000 dilution and 1/200,000 dilution respectively. Blots were developed with Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) secondary antibody at 1/100,000 dilution for 1 hour at room temperature before imaging. The blot was developed on a BIO-RAD^® ChemiDoc^™ MP instrument using the ECL technique.

Exposure time : 3 minutes.

Blocking and Diluting Buffer and concentration : 5% NFDM/TBST.

All lanes:

Western blot - Anti-HDAC4 antibody [EPR22937-157] (ab235583) at 1/1000 dilution

Lane 1:

Wild-type HAP1 whole cell lysate at 20 µg

Lane 2:

HDAC4 knockout HAP1 whole cell 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: 119 kDa

Observed band size: 140 kDa

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• WB

Lab

Western blot - Anti-HDAC4 antibody [EPR22937-157] (AB235583)

Lanes 3-4 in this blot were developed using a higher sensitivity ECL substrate.

Exposure time : Lanes 1-2 : 81 seconds Lane 3 : 48 seconds Lane 4 : 15 seconds.

Blocking and Diluting Buffer and concentration : 5% NFDM/TBST.

All lanes:

Western blot - Anti-HDAC4 antibody [EPR22937-157] (ab235583) at 1/1000 dilution

Lane 1:

HEK-293T (human embryonic kidney epithelial cell), whole cell lysate at 20 µg

Lane 2:

HeLa (human cervix adenocarcinoma epithelial cell), whole cell lysate at 20 µg

Lane 3:

A549 (human lung carcinoma epithelial cell), whole cell lysate at 20 µg

Lane 4:

Neuro-2a (mouse neuroblastoma neuroblast), whole cell 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: 119 kDa

Observed band size: 140 kDa

false

• WB

Lab

Western blot - Anti-HDAC4 antibody [EPR22937-157] (AB235583)

Western blot : Rabbit Monoclonal [EPR22937-157] to HDAC4 ab235583 staining at 1/1000 dilution, shown in green; Mouse anti GAPDH (ab8245) loading control staining at 1/20,000 dilution, shown in magenta.

A band was observed at 137 kDa in Wild-type HCT 116 cell lysates with no signal observed at this size in HDAC4 knockout HCT 116 cell line.

To generate this image, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3pc 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 800CW & Goat anti-Mouse 680RD at 1/20,000 dilution.

All lanes:

Western blot - Anti-HDAC4 antibody [EPR22937-157] (ab235583) at 1/1000 dilution

Lane 1:

Wild-type HCT 116 at 20 µg

Lane 2:

HDAC4 knockout HCT 116 at 20 µg

Lane 2:

Western blot - Human HDAC4 knockout HCT116 cell line (<a href='/en-us/products/cell-lines/human-hdac4-knockout-hct116-cell-line-ab286487'>ab286487</a>) at 20 µg

Lane 3:

Wild-type U-87 MG at 20 µg

Lane 4:

HDAC4 knockout U-87 MG at 20 µg

Lane 5:

Wild-type A549 at 20 µg

Lane 6:

HDAC4 knockout A549 at 20 µg

Secondary

All lanes:

Goat anti-Rabbit 800CW & Goat anti-Mouse 680RD at 1/20000 dilution

Predicted band size: 119 kDa

Observed band size: 137 kDa

false

• WB

Lab

Western blot - Anti-HDAC4 antibody [EPR22937-157] (AB235583)

Exposure time : 3 minutes.

Blocking and Diluting Buffer and concentration : 5% NFDM/TBST.

All lanes:

Western blot - Anti-HDAC4 antibody [EPR22937-157] (ab235583) at 1/1000 dilution

Lane 1:

RAW264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage), 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='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 119 kDa

Observed band size: 140 kDa

false