Recombinant Anti-CBX1 / HP1 beta antibody [EPR24242-78] (ab270988)

Blocking and diluting buffer and concentration: 5% NFDM/TBST

Exposure time: Lanes 1-4: 59 seconds Lanes 5-8: 3 minutes

 Lysates/proteins at 10 µg per lane.

Blocking and diluting buffer and concentration: 5% NFDM/TBST

 Exposure time: Lanes 1-4: 15 seconds Lanes 5-7: 26 seconds

 Lysates/proteins at 20 µg per lane.

Chromatin was prepared from ES-D3 cells according to the Abcam Dual-X-ChIP protocol*. Cells were fixed with 1.5 mM EGS for 30mins and then formaldehyde for 10min.

The ChIP was performed with 25 µg of chromatin, 5 µg of ab270988 (red), or 5 µg of rabbit normal IgG ab172730 (gray) and 25 µl of Protein A/G Dynabeads. The immunoprecipitated DNA was quantified by real time PCR (Sybr green approach). 

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

Blocking and diluting buffer and concentration: Intercept® (TBS) Blocking Buffer diluted with an equal volume of 0.1% TBS

False colour image of Western blot: Anti-CBX1/HP1 beta antibody [EPR24242-78] (ab270988) staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red.

In Western blot, ab270988 was shown to bind specifically to CBX1/HP1. A band was observed at 21 kDa in wild-type HAP1 cell lysates with no signal observed at this size in CBX1/HP1 beta knockout cell line cell. To generate this image, wild-type and CBX1/HP1 beta HAP1 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a PVDF-FL membrane. Membranes were blocked in Odyssey diluted in equal volume of 0.1 % TBS 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 1/10000 dilution.

Performed under reducing conditions. 

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized CBX1 KO HAP1 cells labelling CBX1/HP1 beta with ab270988 at 1/500 dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed antibody at 1/1000 dilution (Green).

Confocal image showing nuclear staining in parental HAP1 cell line, no staining in CBX1 KO HAP1 cell line is observed. ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor^® 594) was used to counterstain tubulin at 1/200 dilution (Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control: Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed at 1/1000 dilution.

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized NIH/3T3 cells labelling CBX1/HP1 beta with ab270988 at 1/500 dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed antibody at 1/1000 dilution (Green). Confocal image showing nuclear staining in NIH/3T3 cell line is observed. ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor^® 594) was used to counterstain tubulin at 1/200 dilution (Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control: Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed at 1/1000 dilution.

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized HeLa cells labelling CBX1/HP1 beta with ab270988 at 1/500 dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed antibody at 1/1000 dilution (Green). Confocal image showing nuclear staining in HeLa cell line is observed. ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor^® 594) was used to counterstain tubulin at 1/200 dilution (Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control: Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed at 1/1000 dilution.

Immunohistochemical analysis of paraffin-embedded Rat testis tissue labelling CBX1/HP1 beta with ab270988 at 1/2000 (0.281 ug/ml) followed by a ready to use Leica DS9800 (BOND^®, Polymer Refine Detection) was used. Nuclear staining on rat testis. The section was incubated with ab270988 for 30 mins 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 Leica DS9800 (BOND^®, Polymer Refine Detection) was used.

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

Immunohistochemical analysis of paraffin-embedded Mouse testis tissue labelling CBX1/HP1 beta with ab270988 at 1/2000 (0.281 ug/ml) followed by a ready to use Leica DS9800 (BOND^®, Polymer Refine Detection) was used. Nuclear staining on mouse testis. (PMID: 32651609) The section was incubated with ab270988 for 30 mins 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 Leica DS9800 (BOND^®, Polymer Refine Detection) was used.

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

Immunohistochemical analysis of paraffin-embedded Human skin tissue labelling CBX1/HP1 beta with ab270988 at 1/2000 (0.281 ug/ml) followed by a ready to use Leica DS9800 (BOND^® , Polymer Refine Detection) was used. Nuclear staining on human skin. (PMID: 25769025) The section was incubated with ab270988 for 30 mins 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 Leica DS9800 (BOND^® , Polymer Refine Detection) was used.

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

Immunohistochemical analysis of paraffin-embedded Human testis tissue labelling CBX1/HP1 beta with ab270988 at 1/2000 (0.281 ug/ml) followed by a ready to use Leica DS9800 (BOND^®, Polymer Refine Detection) was used. Nuclear staining on human testis. The section was incubated with ab270988 for 30 mins 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 Leica DS9800 (BOND^®, Polymer Refine Detection) was used.

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

CBX1/HP1 beta was immunoprecipitated from 0.35 mg NIH/3T3 (mouse embryonic fibroblast), whole cell lysate 10 ug with ab270988 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab270988 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 (Input): NIH/3T3 (mouse embryonic fibroblast), whole cell lysate 10 ug

Lane 2: ab270988 IP in NIH/3T3 whole cell lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab270988 in NIH/3T3 whole cell lysate

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

Exposure time: 15 seconds

CBX1/HP1 beta was immunoprecipitated from 0.35 mg HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate 10ug with ab270988 at 1/30 dilution (2μg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab270988 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate 10ug

Lane 2: ab270988 IP in HeLa whole cell lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab270988 in HeLa whole cell lysate

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

Exposure time: 15 seconds

Flow cytometric analysis of of permeabilized NIH/3T3 (Mouse embryonic fibroblast cells (4% paraformaldehyde fixed, 90% methanol).  Labelling CBX1/HP1 beta with ab270988 at 1/5000 dilution (0.01 ug)(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, ab150081) at 1/2000 dilution was used as the secondary antibody.

Flow cytometric analysis of 4% paraformaldehyde fixed, 90% methanol permeabilized HeLa (human cervix adenocarcinoma epithelial cell) cells. CBX1/HP1 beta with ab270988 at 1/5000 dilution (0.01ug)(Red) was 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, ab150081) at 1/2000 dilution was used as the secondary antibody.

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized Wild-type HAP1 (human chronic myelogenous leukemia near-haploid cell, Right) / CBX1/HP1 beta knockout HAP1(Left) cells labelling CBX1/HP1 beta with ab270988 at 1/5000 dilution (0.01ug)(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, ab150081) at 1/2000 dilution was used as the secondary antibody.