Anti-HDAC2 antibody [3F3]

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-HDAC2 antibody [3F3] (AB51832)

Overlay histogram showing HeLa cells stained with ab51832 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab51832, 1μg/1x10⁶ cells) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 2μg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-HDAC2 antibody [3F3] (AB51832)

ab51832 staining HDAC2 in MCF7 cells. The cells were fixed with 4% paraformaldehyde (10 min), permeabilized with 0.1% PBS-Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated overnight at 4°C with ab51832 at 5µg/ml and ab6046, Rabbit polyclonal to beta Tubulin - Loading Control. Cells were then incubated with ab150117, Goat polyclonal Secondary Antibody to Mouse IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 dilution (shown in green) and ab150080, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor® 594) at 1/1000 dilution (shown in pseudocolour magenta). Nuclear DNA was labelled with DAPI (shown in blue). Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown.

• WB

Lab

Western blot - Anti-HDAC2 antibody [3F3] (AB51832)

Lane 1 : Wild type HAP1 whole cell lysate (20 μg)
Lane 2 : HDAC2 knockout HAP1 whole cell lysate (20 μg)
Lane 3 : A431 whole cell lysate (20 μg)
Lane 4 : HeLa whole cell lysate (20 μg)

Lanes 1 - 4 : Merged signal (red and green). Green - ab51832 observed at 65 kDa. Red - loading control, ab181602, observed at 37 kDa.

ab51832 detected the expected band for HDAC2 in wild-type HAP1 cells and the band was not seen in HDAC2 knockout HAP1 cells. Wild-type and HDAC2 knockout samples were subjected to SDS-PAGE. ab51832 and ab181602 (Rabbit anti GAPDH loading control) were incubated overnight at 4°C at 1000 dilution and 1/10000 dilution respectively. Blots were developed with Goat anti-Mouse IgG H&L (IRDye^® 800CW) preabsorbed ab216772 and Goat anti-Rabbit IgG H&L (IRDye^® 680RD) preabsorbed ab216777 secondary antibodies at 1/10000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-HDAC2 antibody [3F3] (ab51832)

Predicted band size: 55 kDa

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• WB

Lab

Western blot - Anti-HDAC2 antibody [3F3] (AB51832)

This blot was produced using a 4-12% Bis-tris gel under the MOPS buffer system. The gel was run at 200V for 50 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 2% Bovine Serum Albumin before being incubated with ab51832 overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP, and visualised using ECL development solution.

All lanes:

Western blot - Anti-HDAC2 antibody [3F3] (ab51832) at 5 µg/mL

Lane 1:

HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate at 20 µg

Lane 2:

HeLa (Human epithelial carcinoma cell line) Nuclear Lysate at 20 µg

Lane 3:

Jurkat (Human T cell lymphoblast-like cell line) Whole Cell Lysate at 20 µg

Lane 4:

NIH 3T3 (Mouse embryonic fibroblast cell line) Whole Cell Lysate at 20 µg

Lane 5:

K562 (Human erythromyeloblastoid leukemia cell line) Whole Cell Lysate at 20 µg

Lane 6:

U2OS (Human osteosarcoma cell line) Whole Cell Lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Mouse IgG H&L (HRP) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-mouse-igg-h-l-hrp-preadsorbed-ab97040'>ab97040</a>) at 1/10000 dilution

Predicted band size: 55 kDa

Observed band size: 65 kDa

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Exposure time: 16min