Anti-HP1 alpha antibody [EPR5777] - Heterochromatin marker

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-HP1 alpha antibody [EPR5777] - Heterochromatin marker (AB109028)

Immunohistochemical analysis of paraffin-embedded Human tonsil tissue using ab109028 at a dilution of 1/250.

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-HP1 alpha antibody [EPR5777] - Heterochromatin marker (AB109028)

Intracellular Flow Cytometry analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling HP1 alpha with Purified ab109028 at 1/30 dilution (10 μg/mL) (Red). Cells were fixed with 4% Paraformaldehyde and permeabilised with 90% Methanol. A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) secondary antibody was used at 1/2000. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue).

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-HP1 alpha antibody [EPR5777] - Heterochromatin marker (AB109028)

ab109028 staining HP1 alpha in wild-type HAP1 cells (top panel) and HP1 alpha knockout HAP1 cells (bottom panel). The cells were fixed with 4% formaldehyde (10min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab109028 at 1/250 dilution and ab195889 at 1/250 dilution (shown in pseudo colour red) overnight at +4°C, followed by a further incubation at room temperature for 1h with a goat secondary antibody to Rabbit IgG (Alexa Fluor® 488) (ab150081) at 2 μg/ml (shown in green). Nuclear DNA was labelled in blue with DAPI.

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-HP1 alpha antibody [EPR5777] - Heterochromatin marker (AB109028)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human breast cancer tissue sections labeling HP1 alpha with Purified ab109028 at 1/1000 dilution (0.31 µg/mL). Heat mediated antigen retrieval using Bond™ Epitope Retrieval Solution 2 (pH 9.0). Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) was used as the secondary antibody. Negative control : PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-HP1 alpha antibody [EPR5777] - Heterochromatin marker (AB109028)

ab109028 staining HP1 alpha in HeLa (human cervical adenocarcinoma epithelial cell). The cells were fixed with 100% methanol, permeabilized with 0.1% TritonX-100. The cells were then incubated with ab109028 at 1 : 100 dilution. Cells were then incubated with ab150088, Goat Anti-Rabbit IgG H&L (Alexa Fluor® 594) preadsorbed at 1 : 1000 dilution (shown in green). ab195889, Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) was used as counterstain at 1 : 200 dilution (shown in red). Nuclear DNA was labelled with DAPI (shown in blue).

Confocal image showing nuclear staining in HeLa cell line.

• IP

Unknown

Immunoprecipitation - Anti-HP1 alpha antibody [EPR5777] - Heterochromatin marker (AB109028)

Purified ab109028 at 1/30 dilution (2μg) immunoprecipitating HP1 alpha in MCF7 whole cell lysate.
Lane 1 (input) : MCF7 (Human breast adenocarcinoma epithelial cell) whole cell lysate 10μg
Lane 2 (+) : ab109028 + MCF7 whole cell lysate.
Lane 3 (-) : Rabbit monoclonal IgG (ab172730) instead of ab109028 in MCF7 whole cell lysate.
VeriBlot for IP Detection Reagent (HRP) (ab131366) (1/1000 dilution) was used for Western blotting.
Blocking Buffer and concentration : 5% NFDM/TBST.
Diluting buffer and concentration : 5% NFDM/TBST.
Observed band size : 22 kDa

All lanes:

Immunoprecipitation - Anti-HP1 alpha antibody [EPR5777] - Heterochromatin marker (ab109028)

Predicted band size: 22 kDa

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• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-HP1 alpha antibody [EPR5777] - Heterochromatin marker (AB109028)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of mouse kidney tissue sections labeling HP1 alpha with Purified ab109028 at 1/1000 dilution (0.31 µg/mL). Heat mediated antigen retrieval using Bond™ Epitope Retrieval Solution 2 (pH 9.0). Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) was used as the secondary antibody. Negative control : PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-HP1 alpha antibody [EPR5777] - Heterochromatin marker (AB109028)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of rat pancreas tissue sections labeling HP1 alpha with Purified ab109028 at 1/1000 dilution (0.31 µg/mL). Heat mediated antigen retrieval using Bond™ Epitope Retrieval Solution 2 (pH 9.0). Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) was used as the secondary antibody. Negative control : PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

• WB

Unknown

Western blot - Anti-HP1 alpha antibody [EPR5777] - Heterochromatin marker (AB109028)

We are unsure how to define the extra bands.

All lanes:

Western blot - Anti-HP1 alpha antibody [EPR5777] - Heterochromatin marker (ab109028) at 1/1000 dilution

Lane 1:

HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

SH-SY5Y (Human neuroblastoma epithelial cell) whole cell lysate at 20 µg

Lane 3:

Mouse brain lysate at 20 µg

Lane 4:

Rat brain 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 1/20000 dilution

Predicted band size: 22 kDa

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• WB

Lab

Western blot - Anti-HP1 alpha antibody [EPR5777] - Heterochromatin marker (AB109028)

Lane 1 : Wild-type HAP1 cell lysate (20 μg)
Lane 2 : HP1 alpha knockout HAP1 cell lysate (20 μg)
Lane 3 : HeLa cell lysate (20 μg)
Lane 4 : MCF7 cell lysate (20 μg)

Lanes 1 - 4 : Merged signal (red and green). Green - ab109028 observed at 28 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab109028 was shown to specifically react with HP1 alpha when HP1 alpha knockout samples were used. Wild-type and HP1 alpha knockout samples were subjected to SDS-PAGE. ab109028 and ab8245 (loading control to GAPDH) were diluted 1/1000 and 1/20 000 respectively and incubated overnight at 4°C. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1/10000 dilution for 1 h at room temperature before imaging.

All lanes:

Western blot - Anti-HP1 alpha antibody [EPR5777] - Heterochromatin marker (ab109028)

Predicted band size: 22 kDa,24 kDa

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