Anti-KDM5A / Jarid1A / RBBP2 antibody [EPR12742]

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-KDM5A / Jarid1A / RBBP2 antibody [EPR12742] (AB177486)

Intracellular flow cytometric analysis of permeabilized NCCIT cells labeling KDM5A / Jarid1A / RBBP2 with ab177486 at 1/10 dilution (red) or a rabbit IgG (negative) (green).

• IP

Lab

Immunoprecipitation - Anti-KDM5A / Jarid1A / RBBP2 antibody [EPR12742] (AB177486)

KDM5A / Jarid1A / RBBP2 was immunoprecipitated from 0.35 mg NIH/3T3 (mouse embryonic fibroblast) whole cell lysate with ab177486 at 1/30 dilution (2µg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab177486 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 [EPR12742] (ab177486) 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 <a href='/en-us/products/primary-antibodies/kdm5a-jarid1a-rbbp2-antibody-epr18651-ab194286'>ab194286</a> 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 <a href='/en-us/products/primary-antibodies/kdm5a-jarid1a-rbbp2-antibody-epr18651-ab194286'>ab194286</a> 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

Supplier Data

Western blot - Anti-KDM5A / Jarid1A / RBBP2 antibody [EPR12742] (AB177486)

All lanes:

Western blot - Anti-KDM5A / Jarid1A / RBBP2 antibody [EPR12742] (ab177486) at 1/1000 dilution

Lane 1:

HeLa cell lysate at 10 µg

Lane 2:

293T cell lysate at 10 µg

Lane 3:

MCF7 cell lysate at 10 µg

Lane 4:

NCCIT cell lysate at 10 µg

Secondary

All lanes:

Goat anti-rabbit HRP at 1/2000 dilution

Predicted band size: 192 kDa

false

• WB

Lab

Western blot - Anti-KDM5A / Jarid1A / RBBP2 antibody [EPR12742] (AB177486)

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 - ab177486 observed at 240 kDa. Red - loading control, ab18058, observed at 130 kDa.

ab177486 was shown to specifically react with KDM5A in wild-type HAP1 cells. No bands were observed when KDM5A knockout samples were used. Wild-type and KDM5A knockout samples were subjected to SDS-PAGE. ab177486 and ab18058 (Mouse anti Vinculin loading control) were incubated overnight at 4°C at 1/1000 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 [EPR12742] (ab177486)

Predicted band size: 192 kDa

false

• ChIC/CUT&RUN-seq

Lab

ChIC/CUT&RUN sequencing - Anti-KDM5A / Jarid1A / RBBP2 antibody [EPR12742] (AB177486)

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 ab177486 [EPR12742]. 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 [EPR12742] (AB177486)

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 ab177486 [EPR12742]. 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 [EPR12742] (AB177486)

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 ab177486 [EPR12742]. 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.