Anti-HDAC6 antibody [EPR6160]

• WB

Lab

Western blot - Anti-HDAC6 antibody [EPR6160] (AB133539)

False colour image of Western blot : Anti-HDAC6 antibody [EPR6160] 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, ab133539 was shown to bind specifically to HDAC6. A band was observed at 150 kDa in wild-type HeLa cell lysates with no signal observed at this size in HDAC6 CRISPR-Cas9 edited cell line ab264804 (HDAC6 CRISPR-Cas9 edited cell lysate ab257145). The band observed in the CRISPR-Cas9 edited lysate lane above 150 kDa is likely to represent HDAC6 with an insertion. This has not been investigated further and the functional properties of the gene product have not been determined. To generate this image, wild-type and HDAC6 CRISPR-Cas9 edited HeLa 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 (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) at 1/20000 dilution.

All lanes:

Western blot - Anti-HDAC6 antibody [EPR6160] (ab133539) at 1/1000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

HDAC6 CRISPR-Cas9 edited HeLa cell lysate at 20 µg

Predicted band size: 131 kDa

Observed band size: 150 kDa

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• WB

Unknown

Western blot - Anti-HDAC6 antibody [EPR6160] (AB133539)

All lanes:

Western blot - Anti-HDAC6 antibody [EPR6160] (ab133539) at 1/1000 dilution

Lane 1:

293T cell lysate at 10 µg

Lane 2:

HepG2 cell lysate at 10 µg

Lane 3:

K562 cell lysate at 10 µg

Secondary

All lanes:

HRP labelled goat anti-rabbit at 1/2000 dilution

Predicted band size: 131 kDa

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• ChIC/CUT&RUN-seq

Lab

ChIC/CUT&RUN sequencing - Anti-HDAC6 antibody [EPR6160] (AB133539)

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 ab133539 [EPR6160]. 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 [EPR6160] (AB133539)

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 ab133539 [EPR6160]. 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 [EPR6160] (AB133539)

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 ab133539 [EPR6160]. 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.

• OI-RD Scanning

Unknown

OI-RD Scanning - Anti-HDAC6 antibody [EPR6160] (AB133539)

We have systematically measured KD (the equilibrium dissociation constant between the antibody and its antigen), of more than 840 recombinant antibodies to assess not only their individual KD values but also to see the average affinity of antibody. Based on the comparison with published literature values for mouse monoclonal antibodies, Recombinant antibodies appear to be on average 1-2 order of magnitude higher affinity.