Lanes:
Lane 1: Wild type HAP1 whole cell lysate (20 µg)
Lane 2: CBX3 (KO) knockout  HAP1 whole cell lysate (20 µg)
Lane 3: HUVEC whole cell lysate (20 µg)
Lane 4: HepG2 whole cell lysate (20 µg)

Lanes 1 - 4: Merged signal (red and green). Green - ab213167 observed at 23 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab213167 was shown to recognize HP1 gamma/CBX3 when HP1 gamma/CBX3 knockout samples were used, along with additional cross-reactive bands. Wild-type and HP1 gamma/CBX3 knockout samples were subjected to SDS-PAGE. Ab213167 and ab8245 (Mouse anti GAPDH loading control) were incubated overnight at 4°C at 500 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 1/10000 dilution for 1 hour at room temperature before imaging.

All lanes : Anti-HP1 gamma/CBX3 antibody [EPR19803] (ab213167) at 1/1000 dilution

Lane 1 : Human fetal brain lysate
Lane 2 : Human fetal kidney lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG Peroxidase Conjugate, specific to the non-reduced form of IgG at 1/10000 dilution

Predicted band size: 21 kDa
Observed band size: 21 kDa


Exposure time: 3 minutes

All lanes : Anti-HP1 gamma/CBX3 antibody [EPR19803] (ab213167) at 1/1000 dilution

Lane 1 : HepG2 (Human liver hepatocellular carcinoma cell line) whole cell lysate
Lane 2 : HeLa (Human epithelial cell line from cervix adenocarcinoma) 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: 21 kDa
Observed band size: 21 kDa

Blocking/Dilution buffer: 5% NFDM/TBST.

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

All lanes : Anti-HP1 gamma/CBX3 antibody [EPR19803] (ab213167) at 1/1000 dilution

Lane 1 : Mouse brain lysate
Lane 2 : Mouse kidney lysate
Lane 3 : Mouse spleen lysate
Lane 4 : Rat brain lysate
Lane 5 : Rat spleen lysate
Lane 6 : C6 (Rat glial tumor cell line) whole cell lysate
Lane 7 : RAW 264.7 (Mouse macrophage cell line transformed with Abelson murine leukemia virus) whole cell lysate
Lane 8 : PC-12 (Rat adrenal gland pheochromocytoma cell line) whole cell lysate
Lane 9 : NIH/3T3 (Mouse embryonic fibroblast 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: 21 kDa
Observed band size: 21 kDa

Blocking/Dilution buffer: 5% NFDM/TBST.

Exposure time: Lane 1 and 2: 3 minutes; Lane 3: 10 seconds; Lane 4: 30 seconds; Lane 5: 10 seconds; Lane 6,7,8 and 9: 30 seconds.

Immunohistochemical analysis of paraffin-embedded Human colon tissue labeling HP1 gamma/CBX3 with ab213167 at 1/250 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Nucleus staining on epithelial cells of Human colon is observed.

Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is ab97051 at 1/500 dilution.

Perform heat mediated antigen retrieval with EDTA buffer pH 9 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded Human cervix carcinoma tissue labeling HP1 gamma/CBX3 with ab213167 at 1/250 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Nucleus staining on cancer cells of Human cervix carcinoma is observed.

Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is ab97051 at 1/500 dilution.

Perform heat mediated antigen retrieval with EDTA buffer pH 9 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded Mouse testis tissue labeling HP1 gamma/CBX3 with ab213167 at 1/250 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Nucleus staining on Mouse testis is observed.

Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is ab97051 at 1/500 dilution.

Perform heat mediated antigen retrieval with EDTA buffer pH 9 before commencing with IHC staining protocol.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized NIH/3T3 (Mouse embryonic fibroblast cell line) cells labeling HP1 gamma/CBX3 with ab213167 at 1/100 dilution, followed by Goat Anti-Rabbit IgG (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green).

Confocal image showing nuclear staining on NIH/3T3 cells.

Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) at 1/1000 dilution and Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed (ab150120) at 1/1000 dilution (red).

The negative controls are as follows:-

-ve control 1: ab213167 at 1/100 dilution followed by ab150120 at 1/1000 dilution.

-ve control 2: ab7291 at 1/1000 dilution followed by ab150077  at 1/1000 dilution.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (Human epithelial cell line from cervix adenocarcinoma) cells labeling HP1 gamma/CBX3 with ab213167 at 1/100 dilution, followed by Goat Anti-Rabbit IgG (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green).

Confocal image showing nuclear staining on HeLa cells.

The nuclear counterstain is DAPI (blue).

Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) at 1/1000 dilution and Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed (ab150120) at 1/1000 dilution (red).

The negative controls are as follows:-

-ve control 1: ab213167 at 1/100 dilution followed by ab150120 at 1/1000 dilution.

-ve control 2: ab7291  at 1/1000 dilution followed by ab150077  at 1/1000 dilution.

Flow cytometric analysis of 4% paraformaldehyde-fixed HeLa (Human epithelial cell line from cervix adenocarcinoma) cells labeling HP1 gamma/CBX3 with ab213167 at 1/60 dilution (red) compared with a Rabbit IgG, monoclonal-Isotype control (ab172730) (black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (blue). Goat anti rabbit IgG (Alexa Fluor^® 488) at 1/2000 dilution was used as the secondary antibody.

Flow cytometric analysis of 4% paraformaldehyde-fixed NIH/3T3 (Mouse embryonic fibroblast cell line) cells labeling HP1 gamma/CBX3 with ab213167 at 1/60 dilution (red) compared with a Rabbit IgG,monoclonal - Isotype control (ab172730) (black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (blue). Goat anti Rabbit IgG (Alexa Fluor^® 488) at 1/2000 dilution was used as the secondary antibody.