The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
1/1000 - 1/20000. Detects a band of approximately 110 kDa (predicted molecular weight: 100 kDa).
Use at 1-4 µg/mg of lysate.
1/1000 - 1/5000. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
1/500 - 1/2000.
Permeabilization with Triton-X 100 is recommended for formaldehyde-fixed cells.
Use at an assay dependent concentration. PubMed: 21343339
Use at an assay dependent concentration. PubMed: 17542650
Use at an assay dependent concentration. PubMed: 17542650
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.
Immunocytochemistry/Immunofluorescence analysis of formaldelyde-fixed HeLa cells labelling KAP1 with ab10483 at 1/1000 (1μg/ml). A FITC-conjugated goat anti-rabbit IgG (1/100) was used as the secondary antibody.
Western blot - Anti-KAP1 antibody - ChIP Grade (ab10483)
Detection of Human KAP1 by Western Blot and Immunoprecipitation. Samples: A. Whole cell lysate (50 ug for E, 10 ug for T) 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: ab10483 used at the indicated concentration 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 1 minute exposure.
Detection of Human KAP1 by Western Blot and Immunoprecipitation. Samples: A. Whole cell lysate (50 ug for E, 10 ug for T) 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: ab10483 used at the indicated concentration for WB (A) or 2 ug/0.5 mg lysate for IP (B). Immunoprecipitated KAP1 was detected using a KAP1 antibody from another source
Immunoprecipitation - Anti-KAP1 antibody - ChIP Grade (ab10483)This image was taken from an abreview submitted by an Seth Frietze.
The ab10483 antibody was used to immunoprecipitate KAP1 from HEK293 nuclear extracts. Three different amounts of ab10483 or control IgG were tested (1, 2 or 4 ug). The eluates from these experiments as well as the supernatants from the KAP1 IPs were analyzed by Western blotting using the ab10483 antibody. As shown by Western blotting, the presence of a ~110 kDa band demonstrated that KAP1 was specifically precipitated from these extracts. The 50 and 25 kDa bands correspond to the IgG of the IP antibodies.
IHC image of KAP1 staining in human liver carcinoma formalin fixed paraffin embedded tissue section, performed on a Leica BondTM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab10483, 5µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.
For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.