All lanes : Anti-KDM5C / Jarid1C / SMCX + KDM5D / Jarid1D / SMCY antibody [EPR18653] - ChIP Grade (ab194288) at 1/1000 dilution

Lane 1 : Jurkat (Human T cell leukemia cell line from peripheral blood) whole cell lysate
Lane 2 : HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate
Lane 3 : HEL (Human bone marrow erythroleukemia cell line) whole cell lysate
Lane 4 : MCF7 (Human breast adenocarcinoma cell line) 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: 174 kDa
Observed band size: 174 kDa


Exposure time: 3 minutes

Blocking/Dilution buffer: 5% NFDM/TBST.

WB detection of KDM5C + KDM5D in tissue lysates may need optimization. In our hands this antibody could not detect KDM5C + KDM5D in human tissue lysates in WB.

Chromatin was prepared from HeLa (Human epithelial cell line from cervix adenocarcinoma) cells according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10min. The ChIP was performed with 25μg of chromatin, 5μg of ab194288 (blue), and 20μl of Anti-rabbit IgG sepharose beads. 5µg of rabbit normal IgG was added to the beads control (yellow). The immunoprecipitated DNA was quantified by real time PCR (SYBR approach). Primers are located in the first kb of the transcribed region.

KDM5D + KDM5C was immunoprecipitated from 0.35 mg of F9 (Mouse embryonic testicular cancer cell line) whole cell lysate with ab194288 at 1/50 dilution. Western blot was performed from the immunoprecipitate using ab194288 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10000 dilution.

Lane 1: F9 whole cell lysate 10 μg (Input).

Lane 2: ab194288 IP in F9 whole cell lysate.

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab194288 in F9 whole cell lysate.

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

Exposure time: 3 minutes.

Immunohistochemical analysis of paraffin-embedded Human stomach tissue labeling KDM5C / Jarid1C / SMCX+KDM5D / Jarid1D / SMCY with ab194288 at 1/100 dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Nuclear staining on human stomach. The section was incubated with ab194288 for 30mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

All lanes : Anti-KDM5C / Jarid1C / SMCX + KDM5D / Jarid1D / SMCY antibody [EPR18653] - ChIP Grade (ab194288) at 1/5000 dilution

Lane 1 : His-tagged human KDM5D recombinant protein fragment, aa1-200
Lane 2 : His-tagged human KDM5C recombinant protein fragment, aa1-200

Lysates/proteins at 0.01 µg per lane.

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

Predicted band size: 174 kDa


Exposure time: 1 second

Blocking/Dilution buffer: 5% NFDM/TBST.

The predicted molecular weight of KDM5D recombinant protein fragment is 24.5 kDa. The predicted molecular weight of KDM5C recombinant protein fragment is 25 kDa

All lanes : Anti-KDM5C / Jarid1C / SMCX + KDM5D / Jarid1D / SMCY antibody [EPR18653] - ChIP Grade (ab194288) at 1/1000 dilution

Lane 1 : PC-12 (Rat adrenal gland pheochromocytoma cell line) whole cell lysate
Lane 2 : NIH/3T3 (Mouse embryonic fibroblast cell line) whole cell lysate
Lane 3 : F9 (Mouse embryonic testicular cancer cell line) whole cell lysate

Lysates/proteins at 10 µg per lane.

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

Predicted band size: 174 kDa
Observed band size: 174 kDa

Blocking/Dilution buffer: 5% NFDM/TBST.

Exposure time: Lane 1 and 2: 3 minutes; Lane 3: 15 seconds.

WB detection of KDM5C + KDM5D in tissue lysates may need optimization. In our hands this antibody could not detect KDM5C + KDM5D in mouse and rat tissue lysates in WB.

KDM5D + KDM5C was immunoprecipitated from 0.35 mg of HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate with ab194288 at 1/50 dilution. Western blot was performed from the immunoprecipitate using ab194288 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 10 μg (Input).

Lane 2: ab194288 IP in HeLa whole cell lysate.

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab194288 in HeLa whole cell lysate.

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

Exposure time: 3 minutes.

Immunohistochemical analysis of paraffin-embedded Rat stomach tissue labeling KDM5C / Jarid1C / SMCX+KDM5D / Jarid1D / SMCY with ab194288 at 1/1000 dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Nuclear staining on rat stomach. The section was incubated with ab194288 for 30mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

Immunohistochemical analysis of paraffin-embedded Mouse cerebrum tissue labeling KDM5C / Jarid1C / SMCX+KDM5D / Jarid1D / SMCY with ab194288 at 1/1000 dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Nuclear staining on mouse cerebrum. The section was incubated with ab194288 for 30mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.