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 - ab194286 observed at 240 kDa. Red - loading control, ab18058, observed at 130 kDa.

ab194286 was shown to specifically recognize KDM5A in wild-type HAP1 cells along with additional cross-reactive bands. No band was observed when KDM5A knockout samples were examined. Wild-type and KDM5A knockout samples were subjected to SDS-PAGE. Ab194286 and ab18058 (Mouse anti Vinculin loading control) were incubated overnight at 4°C at 1/2500 dilution 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.

 

 

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (Human epithelial cells from cervix adenocarcinoma) cells labeling KDM5A / Jarid1A / RBBP2 with ab194286 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing nuclear staining on HeLa cell line. The nuclear counter stain is DAPI (blue).

Tubulin is detected with Anti-alpha Tubulin mouse MAb (ab7291) at 1/1000 dilution, followed by Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) (ab150120) secondary antibody at 1/1000 dilution (red).

The negative controls are as follows:
-ve control 1: ab194286 at 1/2000 dilution, followed by Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) (ab150120) secondary at 1/1000 dilution.
-ve control 2: Anti-alpha Tubulin mouse MAb (ab7291) at 1/1000 dilution followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary at 1/1000 dilution.

All lanes : Anti-KDM5A / Jarid1A / RBBP2 antibody [EPR18651] (ab194286) at 1/5000 dilution

Lane 1 : LLC (Mouse lung carcinoma) whole cell lysate
Lane 2 : Mouse testis lysate
Lane 3 : HEK-293 (Human epithelial cells from embryonic kidney) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 192 kDa
Observed band size: 192 kDa


Exposure time: 30 seconds

Blocking/Dilution buffer: 5% NFDM/TBST.

Anti-KDM5A / Jarid1A / RBBP2 antibody [EPR18651] (ab194286) at 1/5000 dilution + HeLa (Human epithelial cells from cervix adenocarcinoma) whole cell lysate at 20 µg

Secondary
Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 192 kDa
Observed band size: 192 kDa


Exposure time: 10 seconds

Blocking/Dilution buffer: 5% NFDM/TBST.

Anti-KDM5A / Jarid1A / RBBP2 antibody [EPR18651] (ab194286) at 1/1000 dilution + NIH/3T3 (Mouse embryonic fibroblast cells) whole cell lysate at 10 µg

Secondary
Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 192 kDa
Observed band size: 192 kDa


Exposure time: 10 seconds

Blocking/Dilution buffer: 5% NFDM/TBST.

KDM5A / Jarid1A / RBBP2 was immunoprecipitated from 1mg of HeLa (Human epithelial cells from cervix adenocarcinoma) whole cell lysate with ab194286 at 1/100 dilution. Western blot was performed from the immunoprecipitate using ab194286 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10000 dilution.

Lane 1: HeLa whole cell lysate 10ug (Input).

Lane 2: ab194286 IP in HeLa whole cell lysate.

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab194286 in HeLa whole cell lysate.

Blocking and dilution buffer and concentration: 5% NFDM/TBST.

Exposure time: 5 seconds.

Flow cytometry analysis of NIH/3T3 (mouse embryo) cells labelling KDM5A / Jarid1A / RBBP2 (red) with purified ab194286 at dilution of 1/1500. The secondary antibody used was Alexa Fluor^® 488 goat-anti-rabbit IgG (1/2000). Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. Isotype control antibody used was Rabbit Monoclonal IgG (black). The blue line shows cells without incubation with primary antibody and secondary antibody.