Lane 1: Wild-type HAP1 cell lysate (20 µg)
Lane 2: HDAC4 knockout HAP1 cell lysate (20 µg)
Lane 3: NIH3T3 cell lysate (20 µg)
Lane 4: HeLa cell lysate (20 µg)
Lanes 1 - 4: Merged signal (red and green). Green - ab111318 observed at 140 kDa. Red - loading control, ab8245, observed at 37 kDa.
ab111318 was shown to recognize HDAC4 when HDAC4 knockout samples were used, along with additional cross-reactive bands. Wild-type and HDAC4 knockout samples were subjected to SDS-PAGE. ab111318 and ab8245 (loading control to GAPDH) were both diluted 1 µg/ml and 1/10000 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 1/10000 dilution for 1 hour at room temperature before imaging.

ICC/IF image of ab111318 stained HeLa cells. The cells were 4% formaldehyde fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody ab111318 at 5µg/ml overnight at +4°C. The secondary antibody (green) was DyLight® 488 goat anti- rabbit (ab96899) IgG (H+L) used at a 1/250 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

ab111318 (1/200) staining HDAC4 in HeLa cells (green). Cells were fixed in paraformaldehyde, permeabilised in 0.5% Triton X-100/PBS and counterstained with DAPI in order to highlight the nucleus (red). For further experimental details please refer to Abreview.

See Abreview

All lanes : Anti-HDAC4 antibody (ab111318) at 1 µg/ml (Milk blocking - 3%)

Lane 1 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate
Lane 2 : HEK293 (Human embryonic kidney cell line) Whole Cell Lysate
Lane 3 : U2OS (Human osteosarcoma cell line) Whole Cell Lysate
Lane 4 : HepG2 (Human hepatocellular liver carcinoma cell line) Whole Cell Lysate
Lane 5 : NIH 3T3 (Mouse embryonic fibroblast cell line) Whole Cell Lysate

Lysates/proteins at 25 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) preadsorbed (ab97080) at 1/5000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 119 kDa
Observed band size: 119 kDa


Exposure time: 12 minutes


Abcam recommends using milk as the blocking agent. Abcam welcomes customer feedback and would appreciate any comments regarding this product and the data presented above.