Anti-HDAC6 antibody [EPR1698(2)]

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-HDAC6 antibody [EPR1698(2)] (AB133493)

ab133493 staining HDAC6 in K562 (human chronic myelogenous leukemia) by intracellular flow cytometry. Cells were fixed with 4% paraformaldehyde, permabilised with 90% methanol and the sample was incubated with the primary antibody at a dilution of 1/200. A goat anti rabbit IgG (Alexa Fluorr^® 488) at a dilution of 1/2000 was used as the secondary antibody.

Isoytype control : Rabbit monoclonal IgG (Black)

Unlabelled control : Cell without incubation with primary antibody and secondary antibody (Blue)

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-HDAC6 antibody [EPR1698(2)] (AB133493)

Immunohistochemical analysis of paraffin embedded Human kidney tissue labelling HDAC6 with ab133493 antibody at a dilution of 1/50.

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

• WB

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Western blot - Anti-HDAC6 antibody [EPR1698(2)] (AB133493)

All lanes:

Western blot - Anti-HDAC6 antibody [EPR1698(2)] (ab133493) at 1/10000 dilution

Lane 1:

HeLa cell lysate at 10 µg

Lane 2:

Jurkat cell lysate at 10 µg

Lane 3:

K562 cell lysate at 10 µg

Lane 4:

COS-1 cell lysate at 10 µg

Secondary

All lanes:

HRP labelled goat anti-rabbit at 1/2000 dilution

Predicted band size: 131 kDa

Observed band size: 160 kDa

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• WB

Lab

Western blot - Anti-HDAC6 antibody [EPR1698(2)] (AB133493)

Lanes 1- 2 : Merged signal (red and green). Green - ab133493 observed at 160 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

ab133493 was shown to react with HDAC6 in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line was used. Wild-type HeLa and HDAC6 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. ab133493 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) overnight at 4°C at a 1 in 10000 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-HDAC6 antibody [EPR1698(2)] (ab133493) at 1/10000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

HDAC6 knockout HeLa cell lysate at 20 µg

Predicted band size: 131 kDa

Observed band size: 160 kDa

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• WB

Lab

Western blot - Anti-HDAC6 antibody [EPR1698(2)] (AB133493)

Lanes 1 and 2 : Green signal from target – ab133493 observed at 160 kDa
Lanes 3 and 4 : Red signal from loading control – ab8226 observed at 42 kDa
Lanes 5 and 6 : Merged (red and green) signal

ab133493 was shown to specifically react with HDAC6 when HDAC6 knockout samples were used. Wild-type and HDAC6 knockout samples were subjected to SDS-PAGE. ab133493 and ab8226 (loading control to beta actin) were diluted 1/10 000 and 1/1000 respectively and incubated overnight at 4°C. 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 1/10 000 dilution for 1 h at room temperature before imaging.

Lanes 1 - 2:

Western blot - Anti-HDAC6 antibody [EPR1698(2)] (ab133493) at 1/10000 dilution

Lanes 3 - 4:

Western blot - Anti-HDAC6 antibody [EPR1698(2)] (ab133493) at 1/1000 dilution

Lanes 5 - 6:

Western blot - Anti-HDAC6 antibody [EPR1698(2)] (ab133493)

Lanes 1, 3 and 5:

Wild-type HAP1 cell lysate at 20 µg

Lanes 2 and 4:

HDAC6 knockout HAP1 cell lysate at 20 µg

Lane 6:

HDAC6 knockout HAP1 cell lysate

Predicted band size: 131 kDa

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• ChIC/CUT&RUN-seq

Lab

ChIC/CUT&RUN sequencing - Anti-HDAC6 antibody [EPR1698(2)] (AB133493)

ChIC/CUT&RUN was performed using a pAG-MNase at a final concentration of 700 ng/mL, 2.5 x 10^5 HeLa (human cervix adenocarcinoma epithelial cell) cells and 5 µg of ab133493 [EPR1698(2)]. The resulting DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 10 million reads. The negative IgG control ab172730 is also shown.

The University of Geneva owns patents relevant to ChIC (Chromatin Immuno-Cleavage) methods.

• ChIC/CUT&RUN-seq

Lab

ChIC/CUT&RUN sequencing - Anti-HDAC6 antibody [EPR1698(2)] (AB133493)

ChIC/CUT&RUN was performed using a pAG-MNase at a final concentration of 700 ng/mL, 2.5 x 10^5 HeLa (human cervix adenocarcinoma epithelial cell) cells and 5 µg of ab133493 [EPR1698(2)]. The resulting DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 10 million reads. The negative IgG control ab172730 is also shown.

The University of Geneva owns patents relevant to ChIC (Chromatin Immuno-Cleavage) methods.

• ChIC/CUT&RUN-seq

Lab

ChIC/CUT&RUN sequencing - Anti-HDAC6 antibody [EPR1698(2)] (AB133493)

ChIC/CUT&RUN was performed using a pAG-MNase at a final concentration of 700 ng/mL, 2.5 x 10^5 HeLa (human cervix adenocarcinoma epithelial cell) cells and 5 µg of ab133493 [EPR1698(2)]. The resulting DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 10 million reads. The negative IgG control ab172730 is also shown.

The University of Geneva owns patents relevant to ChIC (Chromatin Immuno-Cleavage) methods.