Human Predicted to work with:
Rabbit, Dog, Chimpanzee, Macaque monkey, Gorilla
Synthetic peptide corresponding to Human KAP1 aa 450-550 (phospho S473) conjugated to Keyhole Limpet Haemocyanin (KLH).
This antibody gave a positive signal within Western Blot in the following whole cell lysates: HeLa; HeLa UV Irradiated; NIH3T3.
This antibody gave a positive result in IF in the following methanol fixed cell line: HeLa.
This antibody gave a positive result in IHC in the following FFPE tissue: Human normal spleen.
Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C or -80°C. Avoid freeze / thaw cycle.
pH: 7.40 Preservative: 0.02% Sodium azide Constituent: PBS Note: Batches of this product that have a concentration < 1mg/ml may have BSA added as a stabilising agent. If you would like information about the formulation of a specific lot, please contact our scientific support team who will be happy to help.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Use a concentration of 1 µg/ml. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
Use a concentration of 1 µg/ml.
Use a concentration of 1 µg/ml. Detects a band of approximately 110 kDa (predicted molecular weight: 88 kDa).
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.
Western blot - Anti-KAP1 (phospho S473) antibody (ab133225)
All lanes : Anti-KAP1 (phospho S473) antibody (ab133225) at 1 µg/ml
Lane 1 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate Lane 2 : Hela Whole Cell Lysate - UV Irradiated Lane 3 : NIH 3T3 (Mouse embryonic fibroblast cell line) Whole Cell Lysate Lane 4 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate with Immunizing peptide at 1 µg/ml Lane 5 : Hela Whole Cell Lysate - UV Irradiated with Immunizing peptide at 1 µg/ml Lane 6 : NIH 3T3 (Mouse embryonic fibroblast cell line) Whole Cell Lysate with Immunizing peptide at 1 µg/ml Lane 7 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate with Control peptide at 1 µg/ml Lane 8 : Hela Whole Cell Lysate - UV Irradiated with Control peptide at 1 µg/ml Lane 9 : NIH 3T3 (Mouse embryonic fibroblast cell line) Whole Cell Lysate with Control peptide at 1 µg/ml
Lysates/proteins at 20 µg per lane.
Secondary Goat Anti-Rabbit IgG H&L (HRP) preadsorbed (ab97080) at 1/5000 dilution Developed using the ECL technique
IHC image of KAP1 (phospho S473) staining in Human normal spleen 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 ab133225, 1µg/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.
ICC/IF image of ab133225 stained HeLa cells. The cells were 100% methanol fixed (5 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 ab133225 at 1µg/ml overnight at +4°C. The secondary antibody (green) was DyLight® 488 goat anti- rabbit (ab96899) IgG (H+L) used at a 1/250 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.