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    products/primary-antibodies/daxx-antibody-e94-ab32140.pdf

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Validated using a knockout cell lineRecombinantRabMAb

Recombinant Anti-Daxx antibody [E94] (ab32140)

  • Datasheet
  • SDS
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Western blot - Anti-Daxx antibody [E94] (ab32140)
  • Immunocytochemistry/ Immunofluorescence - Anti-Daxx antibody [E94] (ab32140)
  • Western blot - Anti-Daxx antibody [E94] (ab32140)
  • Western blot - Anti-Daxx antibody [E94] (ab32140)
  • Flow Cytometry (Intracellular) - Anti-Daxx antibody [E94] (ab32140)
  • Western blot - Anti-Daxx antibody [E94] (ab32140)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Daxx antibody [E94] (ab32140)
  • Anti-Daxx antibody [E94] (ab32140)

Key features and details

  • Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
  • Rabbit monoclonal [E94] to Daxx
  • Suitable for: WB, IHC-P, Flow Cyt (Intra), ICC/IF
  • Knockout validated
  • Reacts with: Mouse, Rat, Human

Conjugates logo Related conjugates and formulations

APC Carrier Free HRP PE

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Overview

  • Product name

    Anti-Daxx antibody [E94]
    See all Daxx primary antibodies
  • Description

    Rabbit monoclonal [E94] to Daxx
  • Host species

    Rabbit
  • Specificity

    Not widely detected in Mouse and Rat
  • Tested applications

    Suitable for: WB, IHC-P, Flow Cyt (Intra), ICC/IFmore details
    Unsuitable for: IP
  • Species reactivity

    Reacts with: Mouse, Rat, Human
  • Immunogen

    Synthetic peptide within Human Daxx aa 250-350. The exact sequence is proprietary.

  • Positive control

    • WB: HeLa and HAP1 cell lysates. IHC-P: Human stomach adenocarcinoma. IF/ICC: HeLa cell line.
  • General notes

    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.

Properties

  • Form

    Liquid
  • Storage instructions

    Shipped at 4°C. Upon delivery aliquot and store at -20°C. Avoid freeze / thaw cycles.
  • Storage buffer

    pH: 7.20
    Preservative: 0.01% Sodium azide
    Constituents: 49% PBS, 50% Glycerol (glycerin, glycerine), 0.05% BSA
  • Concentration information loading...
  • Purity

    Protein A purified
  • Clonality

    Monoclonal
  • Clone number

    E94
  • Isotype

    IgG
  • Research areas

    • Cell Biology
    • Apoptosis
    • Extracellular Signals
    • Death Ligands
    • Microbiology
    • Interspecies Interaction
    • Host Virus Interaction
    • Epigenetics and Nuclear Signaling
    • Chromatin Remodeling
    • Histone chaperones

Associated products

  • Alternative Versions

    • Anti-Daxx antibody [E94] - BSA and Azide free (ab239806)
  • Compatible Secondaries

    • Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077)
  • Isotype control

    • Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730)
  • KO cell lines

    • Human DAXX knockout HeLa cell line (ab265233)
  • KO cell lysates

    • Human DAXX knockout HeLa cell lysate (ab257408)
  • Recombinant Protein

    • Recombinant Human Daxx protein (ab131785)

Applications

The Abpromise guarantee

Our Abpromise guarantee covers the use of ab32140 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
WB
1/5000. Detects a band of approximately 100 kDa (predicted molecular weight: 81 kDa).
IHC-P
Use at an assay dependent concentration. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
Flow Cyt (Intra)
1/100.

ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.

ICC/IF
1/50.
Notes
WB
1/5000. Detects a band of approximately 100 kDa (predicted molecular weight: 81 kDa).
IHC-P
Use at an assay dependent concentration. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
Flow Cyt (Intra)
1/100.

ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.

ICC/IF
1/50.
Application notes
Is unsuitable for IP.

Target

  • Function

    Transcription corepressor known to repress transcriptional potential of several sumoylated transcription factors. Down-regulates basal and activated transcription. Its transcription repressor activity is modulated by recruiting it to subnuclear compartments like the nucleolus or PML/POD/ND10 nuclear bodies through interactions with MCSR1 and PML, respectively. Seems to regulate transcription in PML/POD/ND10 nuclear bodies together with PML and may influence TNFRSF6-dependent apoptosis thereby. Inhibits transcriptional activation of PAX3 and ETS1 through direct protein-protein interactions. Modulates PAX5 activity; the function seems to involve CREBBP. Acts as an adapter protein in a MDM2-DAXX-USP7 complex by regulating the RING-finger E3 ligase MDM2 ubiquitination activity. Under non-stress condition, in association with the deubiquitinating USP7, prevents MDM2 self-ubiquitination and enhances the intrinsic E3 ligase activity of MDM2 towards TP53, thereby promoting TP53 ubiquitination and subsequent proteasomal degradation. Upon DNA damage, its association with MDM2 and USP7 is disrupted, resulting in increased MDM2 autoubiquitination and consequently, MDM2 degradation, which leads to TP53 stabilization. Acts as histone chaperone that facilitates deposition of histone H3.3. Acts as targeting component of the chromatin remodeling complex ATRX:DAXX which has ATP-dependent DNA translocase activity and catalyzes the replication-independent deposition of histone H3.3 in pericentric DNA repeats outside S-phase and telomeres, and the in vitro remodeling of H3.3-containing nucleosomes. Does not affect the ATPase activity of ATRX but alleviates its transcription repression activity. Upon neuronal activation associates with regulatory elements of selected immediate early genes where it promotes deposition of histone H3.3 which may be linked to transcriptional induction of these genes. Required for the recruitment of histone H3.3:H4 dimers to PML-nuclear bodies (PML-NBs); the process is independent of ATRX and facilitated by ASF1A; PML-NBs are suggested to function as regulatory sites for the incorporation of newly synthesized histone H3.3 into chromatin. In case of overexpression of centromeric histone variant CENPA (as found in various tumors) is involved in its mislocalization to chromosomes; the ectopic localization involves a heterotypic tetramer containing CENPA, and histones H3.3 and H4 and decreases binding of CTCF to chromatin. Proposed to mediate activation of the JNK pathway and apoptosis via MAP3K5 in response to signaling from TNFRSF6 and TGFBR2. Interaction with HSPB1/HSP27 may prevent interaction with TNFRSF6 and MAP3K5 and block DAXX-mediated apoptosis. In contrast, in lymphoid cells JNC activation and TNFRSF6-mediated apoptosis may not involve DAXX. Shows restriction activity towards human cytomegalovirus (HCMV).
  • Tissue specificity

    Ubiquitous.
  • Sequence similarities

    Belongs to the DAXX family.
  • Domain

    The Sumo interaction motif mediates Sumo binding, and is required both for sumoylation and binding to sumoylated targets.
  • Post-translational
    modifications

    Sumoylated with SUMO1 on multiple lysine residues.
    Phosphorylated by HIPK1 upon glucose deprivation.
    Polyubiquitinated; which is promoted by CUL3 and SPOP and results in proteasomal degradation. Ubiquitinated by MDM2; inducing its degradation. Deubiquitinated by USP7; leading to stabilize it.
  • Cellular localization

    Nucleus. Diffuse nuclear distribution pattern and no comparable dot-like accumulation of isoform 1 and Cytoplasm. Nucleus, nucleoplasm. Nucleus, PML body. Nucleus, nucleolus. Chromosome, centromere. Dispersed throughout the nucleoplasm, in PML/POD/ND10 nuclear bodies, and in nucleoli (Probable). Colocalizes with histone H3.3, ATRX, HIRA and ASF1A at PML-nuclear bodies (PubMed:12953102, PubMed:14990586, PubMed:23222847, PubMed:24200965). Colocalizes with a subset of interphase centromeres, but is absent from mitotic centromeres (PubMed:9645950). Detected in cytoplasmic punctate structures (PubMed:11842083). Translocates from the nucleus to the cytoplasm upon glucose deprivation or oxidative stress (PubMed:12968034). Colocalizes with RASSF1 in the nucleus (PubMed:18566590). Colocalizes with USP7 in nucleoplasma with accumulation in speckled structures (PubMed:16845383).
  • Target information above from: UniProt accession Q9UER7 The UniProt Consortium
    The Universal Protein Resource (UniProt) in 2010
    Nucleic Acids Res. 38:D142-D148 (2010) .

    Information by UniProt
  • Database links

    • Entrez Gene: 1616 Human
    • Entrez Gene: 13163 Mouse
    • Entrez Gene: 140926 Rat
    • Omim: 603186 Human
    • SwissProt: Q9UER7 Human
    • SwissProt: O35613 Mouse
    • SwissProt: Q8VIB2 Rat
    • Unigene: 336916 Human
    • Unigene: 271809 Mouse
    • Unigene: 870 Rat
    see all
  • Alternative names

    • BING 2 antibody
    • BING2 antibody
    • CENP-C binding protein antibody
    • DAP 6 antibody
    • DAP6 antibody
    • Daxx antibody
    • DAXX_HUMAN antibody
    • Death associated protein 6 antibody
    • Death domain associated protein 6 antibody
    • Death domain associated protein antibody
    • Death domain-associated protein 6 antibody
    • EAP 1 antibody
    • EAP1 antibody
    • ETS1 associated protein 1 antibody
    • ETS1-associated protein 1 antibody
    • Fas binding protein antibody
    • Fas death domain associated protein antibody
    • Fas death domain-associated protein antibody
    • hDaxx antibody
    • MGC126245 antibody
    • MGC126246 antibody
    see all

Images

  • Western blot - Anti-Daxx antibody [E94] (ab32140)
    Western blot - Anti-Daxx antibody [E94] (ab32140)
    All lanes : Anti-Daxx antibody [E94] (ab32140) at 1/5000 dilution

    Lane 1 : Wild-type HeLa cell lysate
    Lane 2 : DAXX knockout HeLa cell lysate

    Lysates/proteins at 20 µg per lane.

    Performed under reducing conditions.

    Predicted band size: 81 kDa
    Observed band size: 100 kDa why is the actual band size different from the predicted?



    Lanes 1- 2: Merged signal (red and green). Green - ab32140 observed at 100 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

     ab32140 was shown to react with Daxx in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line ab265233 (knockout cell lysate ab257408) was used. Wild-type HeLa and DAXX knockout HeLa cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab32140 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) overnight at 4°C at a 1 in 5000 dilution and a 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye®800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye®680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

  • Immunocytochemistry/ Immunofluorescence - Anti-Daxx antibody [E94] (ab32140)
    Immunocytochemistry/ Immunofluorescence - Anti-Daxx antibody [E94] (ab32140)

    ab32140 stained HeLa cells. The cells were 4% formaldehyde fixed for 10 minutes at room temperature and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1hour at room temperature to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab32140 at 10µg/ml) overnight at +4°C. The secondary antibody (pseudo-colored green) was Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed (ab150081) used at a 1/1000 dilution for 1hour at room temperature. Alexa Fluor® 594 WGA was used to label plasma membranes (pseudo-colored red) at a 1/200 dilution for 1hour at room temperature. DAPI was used to stain the cell nuclei (pseudo-colored blue) at a concentration of 1.43µM for 1hour at room temperature.

  • Western blot - Anti-Daxx antibody [E94] (ab32140)
    Western blot - Anti-Daxx antibody [E94] (ab32140)
    All lanes : Anti-Daxx antibody [E94] (ab32140)

    Lane 1 : Wild-type HAP1 whole cell lysate
    Lane 2 : DAXX knockout HAP1 whole cell lysate

    Lysates/proteins at 20 µg per lane.

    Predicted band size: 81 kDa



    Lanes 1 - 2: Merged signal (red and green). Green - ab32140 observed at 100 kDa. Red - loading control, ab9484, observed at 37 kDa.


    ab32140 was shown to specifically react with Daxx in wild-type HAP1 cells as signal was lost in DAXX knockout cells. Wild-type and DAXX knockout samples were subjected to SDS-PAGE. ab32140 and ab9484 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 1/5000 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.

  • Western blot - Anti-Daxx antibody [E94] (ab32140)
    Western blot - Anti-Daxx antibody [E94] (ab32140)
    All lanes : Anti-Daxx antibody [E94] (ab32140) at 0.271 µg/ml

    Lane 1 : PC-12(Rat adrenal gland pheochromocytoma) whole cell lysate
    Lane 2 : NIH/3T3(Mouse embryonic fibroblast) whole cell lysate

    Lysates/proteins at 20 µg per lane.

    Secondary
    All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

    Predicted band size: 81 kDa


    Exposure time: 29 seconds


    Blocking and diluting buffer: 5% NFDM/TBST.

  • Flow Cytometry (Intracellular) - Anti-Daxx antibody [E94] (ab32140)
    Flow Cytometry (Intracellular) - Anti-Daxx antibody [E94] (ab32140)

    Overlay histogram showing HeLa cells stained with ab32140 (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 (ab32140, 1/100 dilution) for 30 min at 22°C. The secondary antibody used was Alexa Fluor® 488 goat anti-rabbit IgG (H&L) (ab150077) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1µg/1x106 cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter.

     

  • Western blot - Anti-Daxx antibody [E94] (ab32140)
    Western blot - Anti-Daxx antibody [E94] (ab32140)
    Anti-Daxx antibody [E94] (ab32140) at 1/5000 dilution + Hela cell lysate

    Predicted band size: 81 kDa
    Observed band size: 100 kDa why is the actual band size different from the predicted?
    Additional bands at: 48 kDa. We are unsure as to the identity of these extra bands.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Daxx antibody [E94] (ab32140)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Daxx antibody [E94] (ab32140)

    Ab32140, at a dilution of 1/50, staining Daxx in paraffin embedded human stomach adenocarcinoma tissue by Immunohistochemistry.

    Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

  • Anti-Daxx antibody [E94] (ab32140)
    Anti-Daxx antibody [E94] (ab32140)

Protocols

To our knowledge, customised protocols are not required for this product. Please try the standard protocols listed below and let us know how you get on.

Click here to view the general protocols

Datasheets and documents

  • SDS download

  • Datasheet download

    Download

References (14)

Publishing research using ab32140? Please let us know so that we can cite the reference in this datasheet.

ab32140 has been referenced in 14 publications.

  • Hartlieb SA  et al. Alternative lengthening of telomeres in childhood neuroblastoma from genome to proteome. Nat Commun 12:1269 (2021). PubMed: 33627664
  • Xiong J  et al. Genomic and Transcriptomic Characterization of Natural Killer T Cell Lymphoma. Cancer Cell 37:403-419.e6 (2020). PubMed: 32183952
  • Aschacher T  et al. Long interspersed element-1 ribonucleoprotein particles protect telomeric ends in alternative lengthening of telomeres dependent cells. Neoplasia 22:61-75 (2020). PubMed: 31846834
  • Wang S  et al. DAXX mediates high phosphate-induced endothelial cell apoptosis in vitro through activating ERK signaling. PeerJ 8:e9203 (2020). PubMed: 32596036
  • Hechtman JF  et al. Performance of DAXX Immunohistochemistry as a Screen for DAXX Mutations in Pancreatic Neuroendocrine Tumors. Pancreas 48:396-399 (2019). PubMed: 30747827
View all Publications for this product

Customer reviews and Q&As

Show All Reviews Q&A
Submit a review Submit a question

Immunohistochemistry (Frozen sections) abreview for Anti-Daxx antibody [E94]

Below Average
Abreviews
Abreviews
abreview image
Application
Immunohistochemistry (Frozen sections)
Sample
Mouse Tissue sections (mouse embryonic spinal cord E11)
Permeabilization
Yes - 0.25%TritonX100
Specification
mouse embryonic spinal cord E11
Blocking step
0.25%TritonX100, 0.2%Gelatine as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 0.2% · Temperature: 25°C
Fixative
Paraformaldehyde
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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Verified customer

Submitted Aug 07 2014

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