All lanes : Anti-KAP1 antibody (ab10484) at 0.1 µg/ml

Lane 1 : HeLa whole cell lysate
Lane 2 : 293T whole cell lysate
Lane 3 : 3T3 whole cell lysate

Lysates/proteins at 50 µg per lane.

Predicted band size: 100 kDa


Exposure time: 1 second

Cells prepared using NETN lysis buffer. Chemiluminescence detection.

ICC/IF image of ab10484 stained HeLa cells. The cells were 4% PFA fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab10484, 1µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human lung carcinoma (left) and ovarian carcinoma (right) tissues labelling KAP1 with ab10484 at 1/5000 (0.2µg/ml) and 1/1000 (1µg/ml). Detection: DAB.

Immunocytochemistry/ Immunofluorescence analysis of U2OS cells labeling KAP1 with ab10484 at 1 μg/ml. Cells were fixed with paraformaldehyde and permeabilized with 0.1% Tween. The cells were blocked with 2% BSA for 45 minutes at 25°C, followed by incubation with ab10484 at 1 μg/ml in PBS for 50 minutes at 25°C. An undiluted monoclonal Goat Anti-rabbbit IgG Alexa Fluor^® 488 was used as the secondary antibody.

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human ovarian carcinoma labeling KAP1 with ab10484 at 1/5000 dilution (0.2 μg/ml). DAB detection, Hematoxylin counterstain.

Detection of Human KAP1 by Western Blot and Immunoprecipitation.

Samples: A. Whole cell lysate (50 ug) from normal 293T cells (E) or 293T cells transiently transfected with a human KAP1 construct (T). B. Whole cell lysate (0.5 mg) from normal 293T cells. Antibodies: ab10484 used at 1 ug/ml for WB (A) or 2 ug/0.5 mg lysate for IP (B). Immunoprecipitated KAP1 was detected using a KAP1 antibody from another source. Detection: Chemiluminescence with a 10 minute (A) or 1 minute (B) exposure.

Ab10484 staining normal human spleen. Staining is localized to the nucleus.
Left panel: with primary antibody at 2 ug/ml. Right panel: isotype control.
Sections were stained using an automated system DAKO Autostainer Plus , at room temperature. Sections were rehydrated and antigen retrieved with the Dako 3-in-1 AR buffer EDTA pH 9.0 in a DAKO PT Link. Slides were peroxidase blocked in 3% H2O2 in methanol for 10 minutes. They were then blocked with Dako Protein block for 10 minutes (containing casein 0.25% in PBS), then incubated with primary antibody for 20 minutes, and detected with Dako Envision Flex amplification kit for 30 minutes. Colorimetric detection was completed with diaminobenzidine for 5 minutes. Slides were counterstained with Haematoxylin and coverslipped under DePeX. Please note that for manual staining we recommend to optimize the primary antibody concentration and incubation time (overnight incubation), and amplification may be required.

ab10484 staining KAP1 in murine coronal brain tissue by Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections). Tissue was fixed with paraformaldehyde and permeabilized using 1% Triton. Samples were then blocked with 5% serum for 1 hour at 25°C followed by incubation with the primary antibody at 2µg/ml for 16 hours at 4°C. An Alexa-Fluor 488-conjugated donkey anti-rabbit polyclonal was used as secondary antibody at a 1/1000 dilution.
Dentate gyrus of the hippocampus immunostained with DAPI (top) and KAP1 (bottom), which nicely labels the nuclei of the entire dentate gyrus.