Chromatin was prepared from human NCCIT cells according to the Abcam Dual-X-ChIP protocol*. Cells were fixed with 1.5 mM EGS for 30mins and then 1% formaldehyde for 10min.

The ChIP was performed with 25 µg of chromatin, 2 µg of ab242149 (red), or 2 µg of rabbit normal IgG ab172730 (gray) and 20 µl of Protein A/G sepharose beads.  The immunoprecipitated DNA was quantified by real time PCR (Taqman approach for active and inactive loci, Sybr green approach for heterochromatic loci).

*https://www.abcam.com/resources?keywords=X%20ChIP%20protocol

Immunohistochemical analysis of paraffin-embedded rat hippocampus tissue labeling CBX4 with ab242149 at 1/200 dilution (2.785 ug/ml) followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP Polymer). Nuclear staining on rat hippocampus (PMID: 28283560). The section was incubated with ab242149 for 30 at RT. The immunostaining staining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP Polymer).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.

CBX4 was immunoprecipitated from 0.35 mg HCT 116 (Human colorectal carcinoma epithelial cell) whole cell lysate with ab242149 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab242149 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366) was used at 1/5000 dilution.

Lane 1: HCT 116 (Human colorectal carcinoma epithelial cell) whole cell lysate 10ug

Lane 2: ab242149 IP in HCT 116 whole cell lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab242149 in HCT 116 whole cell lysate

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

Exposure time: 15 seconds.

 

All lanes : Anti-CBX4 antibody [EPR23053-7] - ChIP Grade (ab242149) at 1/1000 dilution

Lane 1 : HCT 116 (human colorectal carcinoma epithelial cell), whole cell lysate
Lane 2 : RAW 264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage), whole cell lysate
Lane 3 : C6 (rat glial tumor glial cell), 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: 61 kDa
Observed band size: 74 kDa why is the actual band size different from the predicted?

Blocking and dilution buffer: 5% NFDM/TBST.

Exposure time: 26 seconds.

The molecular weight observed is consistent with what has been described in the literature (PMID: 27864346).

Flow cytometric analysis of 4% paraformaldehyde fixed, 90% methanol permeabilized NCCIT (Human pluripotent embryonic carcinoma epithelial cell) cells labelling CBX4 with ab242149 at 1/600 dilution (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

Immunohistochemical analysis of paraffin-embedded Mouse hippocampus tissue labeling CBX4 with ab242149 at 1/200 dilution (2.785 ug/ml) followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP Polymer). Nuclear staining on mouse hippocampus (PMID: 28283560). The section was incubated with ab242149 for 30 at RT. The immunostaining staining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP Polymer).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.

Flow cytometric analysis of 4% paraformaldehyde fixed, 90% methanol permeabilized HCT 116 (Human colorectal carcinoma epithelial cell) cells labelling CBX4 with ab242149 at 1/600 dilution (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

Immunohistochemical analysis of paraffin-embedded Mouse cerebrum tissue labeling CBX4 with ab242149 at 1/200 dilution (2.785 ug/ml) followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP Polymer). Nuclear staining on mouse cerebrum (PMID: 28283560). The section was incubated with ab242149 for 30 mins at RT. The immunostaining staining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP Polymer).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.

All lanes : Anti-CBX4 antibody [EPR23053-7] - ChIP Grade (ab242149) at 1/1000 dilution

Lane 1 : HDLM-2 (human Hodgkin lymphoma) whole cell lysate
Lane 2 : NCCIT (human pluripotent embryonic carcinoma epithelial cell), whole cell lysate
Lane 3 : Mouse thymus tissue lysate
Lane 4 : Rat thymus tissue 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: 61 kDa
Observed band size: 74 kDa why is the actual band size different from the predicted?

Blocking and dilution buffer: 5% NFDM/TBST.

Exposure time: 3 minutes.

The molecular weight observed is consistent with what has been described in the literature (PMID: 27864346).