Lane 1: Wild-type HAP1 whole cell lysate (20 µg)
Lane 2: KDM5A knockout HAP1 whole cell lysate (20 µg)
Lane 3: HeLa whole cell lysate (20 µg)
Lane 4: HEK293 whole cell lysate (20 µg)

Lanes 1 - 4: Merged signal (red and green). Green - ab78322 observed at 240 kDa. Red - loading control, ab176560, observed at 50 kDa.

ab78322 was shown to specifically react with KDM5A in wild type HAP1 cells along with additional cross reactive bands. No bands were observed when KDM5A knockout samples were examined. Ab78322 and ab176560 (Rabbit anti alpha Tubulin loading control) were incubated overnight at 4°C at 1 ug/ml and 1/10000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

 

 

ab78322 staining KDM5A / Jarid1A / RBBP2 [18E8] in HeLa cell line by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with 4 % paraformaldehyde, permeabilized with Triton X-100 0.25% in PBS and blocked with 1.5% BSA for 30 minutes. Samples were incubated with primary antibody (1/200 in PBS + 1% BSA) for overnight at 4°C. An Alexa Fluor^®488-conjugated Goat anti-mouse IgG polyclonal (1/1000 in PBS + 1% BSA) was used as the secondary antibody and incubated for 1 hour at room temperature. DAPI was used to stain the nuclear DNA.

All lanes : Anti-KDM5A / Jarid1A / RBBP2 antibody [18E8] (ab78322) at 1 µg/ml

Lane 1 : HeLa control siRNA
Lane 2 : HeLa RBBP2 siRNA
Lane 3 : Extracts from MCF7 cells
Lane 4 : Extracts from U2OS cells
Lane 5 : Extracts from NIH3T3 cells
Lane 6 : Extracts from J1 (mouse ES) cells

Developed using the ECL technique.

Predicted band size: 196 kDa
Observed band size: 196 kDa

Overlay histogram showing HeLa cells stained with ab78322 (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 (ab78322, 1µg/1x10⁶ cells) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG2a [ICIGG2A] (ab91361, 1µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed.