Anti-KAP1 抗体 [EPR5249] (ab109545)
Key features and details
- Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
- Rabbit monoclonal [EPR5249] to KAP1
- Suitable for: Flow Cyt (Intra), IP, WB, IHC-P, ICC/IF
- Knockout validated
- Reacts with: Human
Related conjugates and formulations
製品の概要
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製品名
Anti-KAP1 antibody [EPR5249]
KAP1 一次抗体 製品一覧 -
製品の詳細
Rabbit monoclonal [EPR5249] to KAP1 -
由来種
Rabbit -
特異性
Although the immunogen is from a phosphor-peptide, this antibody detects phospho and non-phospho KAP1. Based on a peptide blocking experiment it has been found that the signal generated after non-phospho peptide blocking became much weaker, thus indicating that ab109545 shows cross-reactivity with the non-phospho KAP1 at high level. -
アプリケーション
適用あり: Flow Cyt (Intra), IP, WB, IHC-P, ICC/IFmore details -
種交差性
交差種: Human -
免疫原
Synthetic peptide. This information is proprietary to Abcam and/or its suppliers.
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ポジティブ・コントロール
- WB: A431 and MCF7 cell lysates; IHC-P: Human colon and kidney tissue; ICC/IF: Hela cells; IP: A431 cell lysate.
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特記事項
This product is a recombinant monoclonal antibody, which offers several advantages including:
- - High batch-to-batch consistency and reproducibility
- - Improved sensitivity and specificity
- - Long-term security of supply
- - Animal-free production
Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb® patents.
製品の特性
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製品の状態
Liquid -
保存方法
Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C long term. Avoid freeze / thaw cycle. -
バッファー
pH: 7.20
Preservative: 0.05% Sodium azide
Constituents: 0.1% BSA, 40% Glycerol (glycerin, glycerine), 9.85% Tris glycine, 50% Tissue culture supernatant -
Concentration information loading...
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精製度
Protein A purified -
ポリ/モノ
モノクローナル -
クローン名
EPR5249 -
アイソタイプ
IgG -
研究分野
関連製品
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Alternative Versions
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Compatible Secondaries
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Isotype control
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Positive Controls
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Recombinant Protein
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Related Products
アプリケーション
The Abpromise guarantee
Abpromise保証は、 次のテスト済みアプリケーションにおけるab109545の使用に適用されます
アプリケーションノートには、推奨の開始希釈率がありますが、適切な希釈率につきましてはご検討ください。
アプリケーション | Abreviews | 特記事項 |
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Flow Cyt (Intra) |
1/100.
ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody. |
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IP |
1/30.
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WB | (1) |
1/10000 - 1/50000. Detects a band of approximately 110 kDa (predicted molecular weight: 89 kDa).
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IHC-P |
1/100 - 1/250. Perform heat mediated antigen retrieval via the pressure cooker method before commencing with IHC staining protocol. Heat up to 98 degrees C, below boiling, and then let cool for 10-20 min.
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ICC/IF |
1/100 - 1/250.
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特記事項 |
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Flow Cyt (Intra)
1/100. ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody. |
IP
1/30. |
WB
1/10000 - 1/50000. Detects a band of approximately 110 kDa (predicted molecular weight: 89 kDa). |
IHC-P
1/100 - 1/250. Perform heat mediated antigen retrieval via the pressure cooker method before commencing with IHC staining protocol. Heat up to 98 degrees C, below boiling, and then let cool for 10-20 min. |
ICC/IF
1/100 - 1/250. |
ターゲット情報
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機能
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. -
組織特異性
Expressed in all tissues tested including spleen, thymus, prostate, testis, ovary, small intestine, colon and peripheral blood leukocytes. -
パスウェイ
Protein modification; protein sumoylation. -
配列類似性
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. -
ドメイン
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. -
翻訳後修飾
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. -
細胞内局在
Nucleus. Associated with centromeric heterochromatin during cell differentiation through CBX1. - Information by UniProt
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参照データベース
- Entrez Gene: 10155 Human
- Omim: 601742 Human
- SwissProt: Q13263 Human
- Unigene: 467408 Human
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別名
- E3 SUMO protein ligase TRIM28 antibody
- E3 SUMO-protein ligase TRIM28 antibody
- FLJ29029 antibody
see all
画像
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Lane 1: Wild type HAP1 whole cell lysate (20 µg)
Lane 2: HAP1 + DMSO whole cell lysate (20 µg)
Lane 3: HAP1 + Blaomycin whole cell lysate (20 µg)
Lane 4: TRIM28 knockout HAP1 whole cell lysate (20 µg)
Lane 5: TRIM28 knockout HAP1 + DMSO whole cell lysate (20 µg)
Lane 6: TRIM28 knockout HAP1 + Blaomycin whole cell lysate (20 µg)
Lane 7: HeLa + DMSO whole cell lysate (20 µg)
Lane 8: HeLa + Blaomycin whole cell lysate (20 µg)Lanes 1 - 8: Merged signal (red and green). Green - ab109545 observed at 110 kDa. Red - loading control, ab8245, observed at 37 kDa.
ab109545 was shown to specifically react with KAP1 in wild type cells as signal was lost in KAP1 knockout cells. Wild-type and KAP1 knockout samples were subjected to SDS-PAGE. ab109545 and ab8245 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 10000 dilution and 1/20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed ab216776 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.
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ab109545, at a 1/100 dilution, staining KAP1 in HeLa cells
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KAP1 was immunoprecipitated from 0.35 mg A431 (Human epidermoid carcinoma epithelial cell) cell lysate 10 µg with ab109545 at 1/30 dilution (2µg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab109545 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/5000 dilution.
Lane 1: A431 (Human epidermoid carcinoma epithelial cell) cell lysate 10 µg
Lane 2: ab109545 IP in A431 cell lysate
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab109545 in A431 cell lysate
Blocking and dilution buffer and concentration: 5% NFDM/TBST.
Exposure time: 32 seconds
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All lanes : Anti-KAP1 antibody [EPR5249] (ab109545) at 1/10000 dilution
Lane 1 : A431 (Human epidermoid carcinoma epithelial cell) whole cell lysates
Lane 2 : MCF7 (Human breast adenocarcinoma epithelial cell) whole cell lysates
Lysates/proteins at 15 µg per lane.
Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution (Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated)
Developed using the ECL technique.
Predicted band size: 89 kDa
Observed band size: 110 kDa why is the actual band size different from the predicted?
Exposure time: 10 secondsBlocking/diluting buffer and concentration: 5% NFDM/TBST
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ab109545, at a 1/100 dilution, staining KAP1 in formalin-fixed, paraffin-embedded Human colon tissue.
Perform heat mediated antigen retrieval via the pressure cooker method before commencing with IHC staining protocol.
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ab109545, at a 1/100 dilution, staining KAP1 in formalin-fixed, paraffin-embedded Human kidney tissue.
Perform heat mediated antigen retrieval via the pressure cooker method before commencing with IHC staining protocol.
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Overlay histogram showing HeLa cells stained with ab109545 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab190545, 1/100 dilution) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed.
プロトコール
データシートおよび資料
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SDS download
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Datasheet download
参考文献 (3)
ab109545 は 3 報の論文で使用されています。
- Shah PA et al. The Emerging Role of E3 Ubiquitin Ligase SMURF2 in the Regulation of Transcriptional Co-Repressor KAP1 in Untransformed and Cancer Cells and Tissues. Cancers (Basel) 14:N/A (2022). PubMed: 35406379
- Peng Y et al. TRIM28 activates autophagy and promotes cell proliferation in glioblastoma. Onco Targets Ther 12:397-404 (2019). PubMed: 30655676
- Hedström U et al. An Angle on MK2 Inhibition-Optimization and Evaluation of Prevention of Activation Inhibitors. ChemMedChem 14:1701-1709 (2019). PubMed: 31325352