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使用敲除细胞株进行验证重组RabMAb

重组Anti-KAP1抗体[EPR5249] (ab109545)

Reviews (1)Q&A (6)References (5)
Western blot - Anti-KAP1 antibody [EPR5249] (ab109545)
  • Immunocytochemistry/ Immunofluorescence - Anti-KAP1 antibody [EPR5249] (ab109545)
  • Immunoprecipitation - Anti-KAP1 antibody [EPR5249] (ab109545)
  • Western blot - Anti-KAP1 antibody [EPR5249] (ab109545)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-KAP1 antibody [EPR5249] (ab109545)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-KAP1 antibody [EPR5249] (ab109545)
  • Flow Cytometry (Intracellular) - Anti-KAP1 antibody [EPR5249] (ab109545)
  • Anti-KAP1 antibody [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

Conjugates logo Related conjugates and formulations

Carrier Free

概述

  • 产品名称

    Anti-KAP1抗体[EPR5249]
    参阅全部 KAP1 一抗

  • 描述

    兔单克隆抗体[EPR5249] to KAP1

  • 宿主

    Rabbit

  • 特异性

    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.

  • 经测试应用

    适用于: Flow Cyt (Intra), IP, WB, IHC-P, ICC/IFmore details

  • 种属反应性

    与反应: Human

  • 免疫原

    Synthetic peptide. This information is proprietary to Abcam and/or its suppliers.

  • 阳性对照

    • WB: A431 and MCF7 cell lysates; IHC-P: Human colon and kidney tissue; ICC/IF: Hela cells; IP: A431 cell lysate.

  • 常规说明

    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
    For more information see here.

    Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb® patents.

性能

应用

The Abpromise guarantee

Abpromise™承诺保证使用ab109545于以下的经测试应用

“应用说明”部分 下显示的仅为推荐的起始稀释度；实际最佳的稀释度/浓度应由使用者检定。

应用 Ab评论 说明
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)
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.
说明
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.

靶标

  • 功能

    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.

  • 组织特异性

    Expressed in all tissues tested including spleen, thymus, prostate, testis, ovary, small intestine, colon and peripheral blood leukocytes.

  • 通路

    Protein modification; protein sumoylation.

  • 序列相似性

    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.

  • 结构域

    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.

  • 翻译后修饰

    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.

  • 细胞定位

    Nucleus. Associated with centromeric heterochromatin during cell differentiation through CBX1.
  • Information by UniProt

  • 数据库链接

    • 别名

      • E3 SUMO protein ligase TRIM28 antibody
      • E3 SUMO-protein ligase TRIM28 antibody
      • FLJ29029 antibody
      • KAP 1 antibody
      • KAP-1 antibody
      • KRAB associated protein 1 antibody
      • KRAB interacting protein 1 antibody
      • KRAB-associated protein 1 antibody
      • KRAB-interacting protein 1 antibody
      • KRIP 1 antibody
      • KRIP-1 antibody
      • KRIP1 antibody
      • Nuclear corepressor KAP 1 antibody
      • Nuclear corepressor KAP-1 antibody
      • RING finger protein 96 antibody
      • RNF96 antibody
      • TF1B antibody
      • TIF1 beta antibody
      • TIF1-beta antibody
      • TIF1B antibody
      • TIF1B_HUMAN antibody
      • Transcription intermediary factor 1 beta antibody
      • Transcription intermediary factor 1-beta antibody
      • Trim28 antibody
      • Tripartite motif containing 28 antibody
      • tripartite motif containing protein 28 antibody
      • Tripartite motif-containing protein 28 antibody
      see all

    图片

    • Western blot - Anti-KAP1 antibody [EPR5249] (ab109545)
      Western blot - Anti-KAP1 antibody [EPR5249] (ab109545)

      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.

    • Immunocytochemistry/ Immunofluorescence - Anti-KAP1 antibody [EPR5249] (ab109545)
      Immunocytochemistry/ Immunofluorescence - Anti-KAP1 antibody [EPR5249] (ab109545)
      ab109545, at a 1/100 dilution, staining KAP1 in HeLa cells
    • Immunoprecipitation - Anti-KAP1 antibody [EPR5249] (ab109545)
      Immunoprecipitation - Anti-KAP1 antibody [EPR5249] (ab109545)
      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

    • Western blot - Anti-KAP1 antibody [EPR5249] (ab109545)
      Western blot - Anti-KAP1 antibody [EPR5249] (ab109545)
      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 seconds


      Blocking/diluting buffer and concentration: 5% NFDM/TBST

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-KAP1 antibody [EPR5249] (ab109545)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-KAP1 antibody [EPR5249] (ab109545)

      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.

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-KAP1 antibody [EPR5249] (ab109545)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-KAP1 antibody [EPR5249] (ab109545)

      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.

    • Flow Cytometry (Intracellular) - Anti-KAP1 antibody [EPR5249] (ab109545)
      Flow Cytometry (Intracellular) - Anti-KAP1 antibody [EPR5249] (ab109545)

      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.

       

    • Anti-KAP1 antibody [EPR5249] (ab109545)
      Anti-KAP1 antibody [EPR5249] (ab109545)

    文献 (5)

    发表研究结果有使用 ab109545？请让我们知道，以便我们可以引用本数据表中的参考文章。

    ab109545 被引用在 5 文献中.

    • Turchi L  et al. CELF2 Sustains a Proliferating/OLIG2+ Glioblastoma Cell Phenotype via the Epigenetic Repression of SOX3. Cancers (Basel) 15:N/A (2023). PubMed: 37894405
    • 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
    • Ni X  et al. Role of salt‑inducible kinase 2 in the malignant behavior and glycolysis of colorectal cancer cells. Mol Med Rep 24:N/A (2021). PubMed: 34558647
    • 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

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    Western blot abreview for Anti-KAP1 (phospho S473) antibody [EPR5249]

     Below Average
    Abreviews
    abreview image
    Application
    Western blot
    Sample
    Human Cell lysate - whole cell (Hela cells)
    Loading amount
    50 µg
    Specification
    Hela cells
    Treatment
    non treated, AZD7762 for 4 hrs, etoposide 2µM for 3 hrs, 50 nM AZD7762 for 1h+ etoposide 3h
    Gel Running Conditions
    Reduced Denaturing (8%)
    Blocking step
    BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 18°C
    Read More
    The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

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    提交于 May 15 2012

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