Anti-CBX4 antibody [EPR23053-7] - ChIP Grade

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-CBX4 antibody [EPR23053-7] - ChIP Grade (AB242149)

Intracellular 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.

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-CBX4 antibody [EPR23053-7] - ChIP Grade (AB242149)

Intracellular 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.

• IP

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Immunoprecipitation - Anti-CBX4 antibody [EPR23053-7] - ChIP Grade (AB242149)

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:

Immunoprecipitation - Anti-CBX4 antibody [EPR23053-7] - ChIP Grade (ab242149)

Predicted band size: 52 kDa,61 kDa

Observed band size: 45 kDa,60 kDa,66 kDa,74 kDa

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• ChIP

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ChIP - Anti-CBX4 antibody [EPR23053-7] - ChIP Grade (AB242149)

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).

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CBX4 antibody [EPR23053-7] - ChIP Grade (AB242149)

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.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CBX4 antibody [EPR23053-7] - ChIP Grade (AB242149)

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.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CBX4 antibody [EPR23053-7] - ChIP Grade (AB242149)

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.

• WB

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Western blot - Anti-CBX4 antibody [EPR23053-7] - ChIP Grade (AB242149)

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).

All lanes:

Western blot - Anti-CBX4 antibody [EPR23053-7] - ChIP Grade (ab242149) at 1/1000 dilution

Lane 1:

HDLM-2 (human Hodgkin lymphoma) whole cell lysate at 20 µg

Lane 2:

NCCIT (human pluripotent embryonic carcinoma epithelial cell), whole cell lysate at 20 µg

Lane 3:

Mouse thymus tissue lysate at 20 µg

Lane 4:

Rat thymus tissue 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: 61 kDa

Observed band size: 74 kDa

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• WB

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Western blot - Anti-CBX4 antibody [EPR23053-7] - ChIP Grade (AB242149)

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).

All lanes:

Western blot - Anti-CBX4 antibody [EPR23053-7] - ChIP Grade (ab242149) at 1/1000 dilution

Lane 1:

HCT 116 (human colorectal carcinoma epithelial cell), whole cell lysate at 20 µg

Lane 2:

RAW 264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage), whole cell lysate at 20 µg

Lane 3:

C6 (rat glial tumor glial cell), 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: 61 kDa

Observed band size: 74 kDa

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• WB

Lab

Western blot - Anti-CBX4 antibody [EPR23053-7] - ChIP Grade (AB242149)

False colour image of Western blot : Anti-CBX4 antibody [EPR23053-7] - ChIP Grade staining at 1/1000 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab242149 was shown to bind specifically to CBX4. A band was observed at 75 kDa in wild-type HEK-293 cell lysates with no signal observed at this size in Cbx4 knockout cell line ab261723 (knockout cell lysate ab261666). The band observed in the knockout lysate lane below 75 kDa is likely to represent a truncated form of CBX4. This has not been investigated further and the functional properties of the gene product have not been determined. To generate this image, wild-type and Cbx4 knockout HEK-293 cell lysates were analysed.First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3% milk in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4°C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (ab216776) at 1/20000 dilution.

All lanes:

Western blot - Anti-CBX4 antibody [EPR23053-7] - ChIP Grade (ab242149) at 1/1000 dilution

Lane 1:

Wild-type HEK-293 cell lysate at 20 µg

Lane 2:

Cbx4 knockout HEK-293 cell lysate at 20 µg

Lane 3:

RAW 264.7 cell lysate at 20 µg

Lane 4:

HUVEC cell lysate at 20 µg

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• WB

Lab

Western blot - Anti-CBX4 antibody [EPR23053-7] - ChIP Grade (AB242149)

False colour image of Western blot : Anti-CBX4 antibody [EPR23053-7] - ChIP Grade staining at 1/1000 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab242149 was shown to bind specifically to CBX4. A band was observed at 75 kDa in wild-type HEK-293 cell lysates with no signal observed at this size in Cbx4 CRISPR-Cas9 edited cell line ab261723 (CRISPR-Cas9 edited cell lysate ab261666). The band observed in the CRISPR-Cas9 edited lysate lane below 75 kDa is likely to represent a truncated form of CBX4. This has not been investigated further and the functional properties of the gene product have not been determined. To generate this image, wild-type and Cbx4 CRISPR-Cas9 edited HEK-293 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3% milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4°C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) at 1/20000 dilution.

All lanes:

Western blot - Anti-CBX4 antibody [EPR23053-7] - ChIP Grade (ab242149) at 1/1000 dilution

Lane 1:

Wild-type HEK-293 cell lysate at 20 µg

Lane 2:

Cbx4 CRISPR-Cas9 edited HEK-293 cell lysate at 20 µg

Lane 2:

Western blot - Human CBX4 knockout HEK-293 cell line (<a href='/en-us/products/cell-lines/human-cbx4-knockout-hek-293-cell-line-ab261723'>ab261723</a>)

Lane 3:

RAW 264.7 cell lysate at 20 µg

Lane 4:

HUVEC cell lysate at 20 µg

Predicted band size: 61 kDa

Observed band size: 75 kDa

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