Anti-KDM5A / Jarid1A / RBBP2 antibody [EPR18651]

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-KDM5A / Jarid1A / RBBP2 antibody [EPR18651] (AB194286)

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.

• IP

Supplier Data

Immunoprecipitation - Anti-KDM5A / Jarid1A / RBBP2 antibody [EPR18651] (AB194286)

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.

All lanes:

Immunoprecipitation - Anti-KDM5A / Jarid1A / RBBP2 antibody [EPR18651] (ab194286)

Predicted band size: 192 kDa

false

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-KDM5A / Jarid1A / RBBP2 antibody [EPR18651] (AB194286)

Intracellular 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 Fluorr^® 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.

• IP

Lab

Immunoprecipitation - Anti-KDM5A / Jarid1A / RBBP2 antibody [EPR18651] (AB194286)

KDM5A / Jarid1A / RBBP2 was immunoprecipitated from 0.35 mg NIH/3T3 (mouse embryonic fibroblast) whole cell lysate with ab194286 at 1/30 dilution (2µg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab194286 at 1/1000 dilution.

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

This blot was developed using a high-sensitivity ECL substrate, allowing for the detection of proteins in the mid-femtogram range.

All lanes:

Immunoprecipitation - Anti-KDM5A / Jarid1A / RBBP2 antibody [EPR18651] (ab194286) at 1/30 dilution

Lane 1:

3T3-L1 (mouse embryonic fibroblast) transfected with scrambled siRNA control whole cell lysate at 7 µg

Lane 2:

3T3-L1 transfected with scrambled siRNA control whole cell lysate

Lane 3:

Rabbit monoclonal IgG (<a href='/en-us/products/primary-antibodies/rabbit-igg-monoclonal-epr25a-isotype-control-ab172730'>ab172730</a>) instead of ab194286 in 3T3-L1 transfected with scrambled siRNA control lysate

Lane 4:

3T3-L1 transfected with siRNA specifically targeting KDM5A whole cell lysate at 7 µg

Lane 5:

3T3-L1 transfected with siRNA specifically targeting KDM5A whole cell lysate

Lane 6:

Rabbit monoclonal IgG (<a href='/en-us/products/primary-antibodies/rabbit-igg-monoclonal-epr25a-isotype-control-ab172730'>ab172730</a>) instead of ab194286 in 3T3-L1 transfected with siRNA specifically targeting KDM5A lysate

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/5000 dilution

Observed band size: 200 kDa

true

Exposure time: 180s

• WB

Lab

Western blot - Anti-KDM5A / Jarid1A / RBBP2 antibody [EPR18651] (AB194286)

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.

All lanes:

Western blot - Anti-KDM5A / Jarid1A / RBBP2 antibody [EPR18651] (ab194286)

Predicted band size: 192 kDa

false

• WB

Supplier Data

Western blot - Anti-KDM5A / Jarid1A / RBBP2 antibody [EPR18651] (AB194286)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-KDM5A / Jarid1A / RBBP2 antibody [EPR18651] (ab194286) at 1/5000 dilution

Lane 1:

LLC (Mouse lung carcinoma) whole cell lysate at 20 µg

Lane 2:

Mouse testis lysate at 20 µg

Lane 3:

HEK-293 (Human epithelial cells from embryonic kidney) whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 192 kDa

Observed band size: 192 kDa

false

Exposure time: 30s

• WB

Supplier Data

Western blot - Anti-KDM5A / Jarid1A / RBBP2 antibody [EPR18651] (AB194286)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-KDM5A / Jarid1A / RBBP2 antibody [EPR18651] (ab194286) at 1/5000 dilution

All lanes:

HeLa (Human epithelial cells from cervix adenocarcinoma) whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 161 kDa,192 kDa,22 kDa,40 kDa,43 kDa,46 kDa

Observed band size: 162 kDa,192 kDa,43 kDa,46 kDa

false

Exposure time: 10s

• ChIC/CUT&RUN-seq

Lab

ChIC/CUT&RUN sequencing - Anti-KDM5A / Jarid1A / RBBP2 antibody [EPR18651] (AB194286)

ChIC/CUT&RUN was performed using a pAG-MNase at a final concentration of 700 ng/mL, 2.5 x 10^5 K-562 (human chronic myelogenous leukemia lymphoblast) cells and 5 µg of ab194286 [EPR18651]. 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-KDM5A / Jarid1A / RBBP2 antibody [EPR18651] (AB194286)

ChIC/CUT&RUN was performed using a pAG-MNase at a final concentration of 700 ng/mL, 2.5 x 10^5 K-562 (human chronic myelogenous leukemia lymphoblast) cells and 5 µg of ab194286 [EPR18651]. 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-KDM5A / Jarid1A / RBBP2 antibody [EPR18651] (AB194286)

ChIC/CUT&RUN was performed using a pAG-MNase at a final concentration of 700 ng/mL, 2.5 x 10^5 K-562 (human chronic myelogenous leukemia lymphoblast) cells and 5 µg of ab194286 [EPR18651]. 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.

• WB

Supplier Data

Western blot - Anti-KDM5A / Jarid1A / RBBP2 antibody [EPR18651] (AB194286)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-KDM5A / Jarid1A / RBBP2 antibody [EPR18651] (ab194286) at 1/1000 dilution

All lanes:

NIH/3T3 (Mouse embryonic fibroblast cells) whole cell lysate at 10 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 192 kDa

Observed band size: 192 kDa

false

Exposure time: 10s