The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
1/500 - 1/10000. Detects a band of approximately 110 kDa (predicted molecular weight: 100 kDa).
Use at 2-10 µg/mg of lysate.
Use a concentration of 1 µg/ml.
Use a concentration of 2 µg/ml.
Nuclear corepressor for KRAB domain-containing zinc finger proteins (KRAB-ZFPs). Mediates gene silencing by recruiting CHD3, a subunit of the nucleosome remodeling and deacetylation (NuRD) complex, and SETDB1 (which specifically methylates histone H3 at 'Lys-9' (H3K9me)) to the promoter regions of KRAB target genes. Enhances transcriptional repression by coordinating the increase in H3K9me, the decrease in histone H3 'Lys-9 and 'Lys-14' acetylation (H3K9ac and H3K14ac, respectively) and the disposition of HP1 proteins to silence gene expression. Recruitment of SETDB1 induces heterochromatinization. May play a role as a coactivator for CEBPB and NR3C1 in the transcriptional activation of ORM1. Also corepressor for ERBB4. Inhibits E2F1 activity by stimulating E2F1-HDAC1 complex formation and inhibiting E2F1 acetylation. May serve as a partial backup to prevent E2F1-mediated apoptosis in the absence of RB1. Important regulator of CDKN1A/p21(CIP1). Has E3 SUMO-protein ligase activity toward itself via its PHD-type zinc finger.
Expressed in all tissues tested including spleen, thymus, prostate, testis, ovary, small intestine, colon and peripheral blood leukocytes.
Protein modification; protein sumoylation.
Belongs to the TRIM/RBCC family. Contains 2 B box-type zinc fingers. Contains 1 bromo domain. Contains 1 PHD-type zinc finger. Contains 1 RING-type zinc finger.
The HP1 box is both necessary and sufficient for HP1 binding. The PHD-type zinc finger enhances CEBPB transcriptional activity. The PHD-type zinc finger, the HP1 box and the bromo domain, function together to assemble the machinery required for repression of KRAB domain-containing proteins. Acts as an intramolecular SUMO E3 ligase for autosumoylation of bromodomain. The RING-finger-B Box-coiled-coil/tripartite motif (RBCC/TRIM motif) is required for interaction with the KRAB domain of KRAB-zinc finger proteins. Binds four zinc ions per molecule. The RING finger and the N-terminal of the leucine zipper alpha helical coiled-coil region of RBCC are required for oligomerization. Contains one Pro-Xaa-Val-Xaa-Leu (PxVxL) motif, which is required for interaction with chromoshadow domains. This motif requires additional residues -7, -6, +4 and +5 of the central Val which contact the chromoshadow domain.
Phosphorylated upon DNA damage, probably by ATM or ATR. ATM-induced phosphorylation on Ser-824 represses sumoylation leading to the de-repression of expression of a subset of genes involved in cell cycle control and apoptosis in response to genotoxic stress. Dephosphorylation by the phosphatases, PPP1CA and PP1CB forms, allows sumoylation and expression of TRIM28 target genes. Sumoylation/desumoylation events regulate TRIM28-mediated transcriptional repression. Sumoylation is required for interaction with CHD3 and SETDB1 and the corepressor activity. Represses and is repressed by Ser-824 phosphorylation. Enhances the TRIM28 corepressor activity, inhibiting transcriptional activity of a number of genes including GADD45A and CDKN1A/p21. Lys-554, Lys-779 and Lys-804 are the major sites of sumoylation. In response to Dox-induced DNA damage, enhanced phosphorylation on Ser-824 prevents sumoylation and allows de-repression of CDKN1A/p21.
Nucleus. Associated with centromeric heterochromatin during cell differentiation through CBX1.
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 - Anti-KAP1 antibody (ab10484)This image is courtesy of an anonymous abreview.
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.
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.
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-KAP1 antibody (ab10484)Image courtesy of an anonymous Abreview.
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.