Anti-HP1 gamma antibody (ab10480)

Immunocytochemistry/ Immunofluorescence - HP1 gamma antibody (ab10480)

Immunofluorescent imaging of human cells (U2OS) with ab10480 reveals the expected exclusively nuclear staining,  corresponding to the known nuclear localisation of HP1-gamma.

IF was performed with a standard paraformaldehyde technique (fixed in PBS buffered PFH 4% for 5 minutes, permeabilised with 0.5% triton-PBS for 5 minutes, blocked with 5% milk / 0.2% tween for one hour.  Primary antibody used at 1/200 in 5% milk / 0.2% TWEEN for one hour, secondary antibody for 30 minutes.  All blocking and incubation steps carried out at 37 degrees C. Nuclei stained with Hoechst stain (blue).

Immunofluorescent imaging of human cells (U2OS) with ab10480 reveals the expected exclusively nuclear staining, corresponding to the known nuclear localisation of HP1-gamma. IF was performed with a standard paraformaldehyde technique (fixed in PBS buffered PFH 4% for 5 minutes, permeabilised with 0.5% triton-PBS for 5 minutes, blocked with 5% milk / 0.2% tween for one hour. Primary antibody used at 1/200 in 5% milk / 0.2% TWEEN for one hour, secondary antibody for 30 minutes. All blocking and incubation steps carried out at 37 degrees C. Nuclei stained with Hoechst stain (blue).

Luke Hughes-Davies and Rhiannon Jade, Gurdon Institute, Cambridge, UK

Immunofluorescence - HP1 gamma antibody (ab10480)

ab10480 was used to stain HeLa cells in interphase in panel one. In the second panel, the cell is stained by ab10480 (green), DAPI (blue) and SH-CREST (red) which stains the centromeres.

Fix 30 minutes on ice in 4% formaldehyde in PEM. Quench autofluorescence 2 x 5 minutes with 1 mg/ml Na borohydride or 100 mM ammonium chloride in PEM. Permeablize 30 minutes with 0.5% TX-100 in PEM. Block 30 minutes in 5% milk in TBST. Primary antibody incubated overnight at 4oC diluted 1/100 in 5% milk in TBST. Secondary antibody incubaged 1 hour at RT diluted in 5% milk in TBST. Post-fix 20 minutes on ice in 4% formaldehyde in PEM. Quench autofluorescence 2 x 5 minutes with ammonium chloride in PEM. Counterstain with DAPI in TBST. Mount with ProLong Gold antifade reagent from Invitrogen. Notes: Ample washing between each step. TBST = Tris buffered saline + 0.1% Tween. PEM = 80 mM K-PIPES, pH 6.8, 5 mM EGTA, 2 mM MgCl2.

Scott Slattery and Mike Mancini

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - HP1 gamma antibody (ab10480)

ab10480 at a 1/200 dilution staining HP1 gamma in human skin epidermis tissue by Immunohistochemistry (Formalin fixed, paraffin embedded) incubated for 20 hours at 4°C. Heat mediated antigen retrieval step performed using citrate buffer pH 6.0, microwave 10 minutes. Permeabilized using Tris-Buffered Saline Tween-20. Blocked using 2% BSA for 1 hour at 22°C. Secondary used at a 1/200 dilution Goat polyclonal to Rabbit IgG - H&L (FITC) (ab6717). The nuclei are counterstained with propidium iodide (red).

This image is courtesy of an anonymous abreview.

Western blot - HP1 gamma antibody (ab10480)

All lanes : Anti-HP1 gamma antibody (ab10480) at 1 µg/ml

Lane 1 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate
Lane 2 : MCF7 (Human breast adenocarcinoma cell line) Whole Cell Lysate
Lane 3 : HEK293 (Human embryonic kidney cell line) Whole Cell Lysate

Lysates/proteins at 10 µg per lane.

Secondary
Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (HRP), pre-adsorbed (ab97080) at 1/5000 dilution
developed using the ECL technique

Performed under reducing conditions.

Predicted band size : 21 kDa
Observed band size : 24 kDa (why is the actual band size different from the predicted?)
Additional bands at : 49 kDa. We are unsure as to the identity of these extra bands.

Exposure time : 12 minutes

Western blot - Anti-HP1 gamma antibody (ab10480)

Anti-HP1 gamma antibody (ab10480) at 1/500 dilution + HP1 gamma protein (Human) (ab88011) at 0.01 µg

Secondary
Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (HRP), pre-adsorbed (ab97080) at 1/5000 dilution
developed using the ECL technique

Performed under reducing conditions.

Exposure time : 30 seconds

Ab10480 recognizes the tagged recombinant HP1 gamma protein (ab88011) which has an expected molecular weight of ~50 kDa.