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95830
ATRX/Daxx Antibody Sampler Kit
Primary Antibodies
Antibody Sampler Kit
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ATRX/Daxx Antibody Sampler Kit #95830

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ATRX/Daxx Antibody Sampler Kit: Image 1

Chromatin immunoprecipitations were performed with cross-linked chromatin from HeLa cells and either ATRX (D1N2E) Rabbit mAb or Normal Rabbit IgG #2729, using SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003. The enriched DNA was quantified by real-time PCR using human 18s rDNA repeat primers, SimpleChIP® Human 28s rDNA Repeat Primers #14901, and SimpleChIP® Human GAPDH Exon 1 Primers #5516. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin, which is equivalent to one.

ATRX/Daxx Antibody Sampler Kit: Image 2

Confocal immunofluorescent analysis of HeLa cells using Daxx (25C12) Rabbit mAb (green). Actin filaments have been labeled with Alexa Fluor® 555 phalloidin (red).

ATRX/Daxx Antibody Sampler Kit: Image 3

Flow cytometric analysis of human peripheral blood lymphocytes using Histone H3 (D1H2) XP® Rabbit mAb (blue) compared to Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (red). Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 647 Conjugate) #4414 was used as a secondary antibody.

ATRX/Daxx Antibody Sampler Kit: Image 4

After the primary antibody is bound to the target protein, a complex with HRP-linked secondary antibody is formed. The LumiGLO® is added and emits light during enzyme catalyzed decomposition.

ATRX/Daxx Antibody Sampler Kit: Image 5

Immunoprecipitation of ATRX from 293T cell extracts using Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (lane 2) or ATRX (D1N2E) Rabbit mAb (lane 3). Lane 1 is 10% input. Western blot analysis was performed using ATRX (D1N2E) Rabbit mAb.

ATRX/Daxx Antibody Sampler Kit: Image 6

Western blot analysis of extracts from K562 (human), A20 (mouse) and PC12 (rat) cell lines using Daxx (25C12) Rabbit mAb.

ATRX/Daxx Antibody Sampler Kit: Image 7

Chromatin immunoprecipitations were performed with cross-linked chromatin from HeLa cells and either Tri-Methyl-Histone H3 (Lys9) (D4W1U) Rabbit mAb or Normal Rabbit IgG #2729 using SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003. The enriched DNA was quantified by real-time PCR using SimpleChIP® Human GAPDH Exon 1 Primers #5516, SimpleChIP® Human AFM Intron 1 Primers #5098, SimpleChIP® Human MYT-1 Exon 1 Primers #4493, and SimpleChIP® Human α Satellite Repeat Primers #4486. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin, which is equivalent to one.

ATRX/Daxx Antibody Sampler Kit: Image 8

Flow cytometric analysis of HeLa cells using Tri-Methyl-Histone H3 (Lys9) (D4W1U) Rabbit mAb (solid line) compared to concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (dashed line). Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.

ATRX/Daxx Antibody Sampler Kit: Image 9

Confocal immunofluorescent analysis of HeLa cells using Histone H3 (D1H2) XP® Rabbit mAb (green) and β-Tubulin (9F3) Rabbit mAb (Alexa Fluor® 555 Conjugate) #2116 (red).

ATRX/Daxx Antibody Sampler Kit: Image 10

Western blot analysis of extracts from various cell lines using ATRX (D1N2E) Rabbit mAb (upper) and Nucleolin (D4C7O) Rabbit mAb #14574. As expected, the signal for ATRX is not present in the negative cell line U2OS.

ATRX/Daxx Antibody Sampler Kit: Image 11

Confocal immunofluorescent analysis of interphase (left) or mitotic (right) HeLa cells, untreated (upper) or λ phosphatase-treated (lower), using Tri-Methyl-Histone H3 (Lys9) (D4W1U) Rabbit mAb (green) and β-Actin (8H10D10) Mouse mAb #3700 (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye). As shown, this antibody does not detect tri-methyl histone H3 Lys9 in mitotic cells when the adjacent Ser10 residue is phosphorylated.

ATRX/Daxx Antibody Sampler Kit: Image 12

Immunohistochemical analysis of paraffin-embedded human breast carcinoma using Histone H3 (D1H2) XP® Rabbit mAb.

ATRX/Daxx Antibody Sampler Kit: Image 13

Western blot analysis of extracts from various cell lines using Tri-Methyl-Histone H3 (Lys9) (D4W1U) Rabbit mAb.

ATRX/Daxx Antibody Sampler Kit: Image 14

Western blot analysis of extracts from various cell lines using Histone H3 (D1H2) XP® Rabbit mAb.

ATRX/Daxx Antibody Sampler Kit: Image 15

Antibody specificity was determined by western blotting. HeLa and NIH/3T3 cell lysates were probed with Tri-Methyl-Histone H3 (Lys9) (D4W1U) Rabbit mAb (Panel A) or Tri-Methyl-Histone H3 (Lys9) (D4W1U) Rabbit mAb pre-adsorbed with 1.5 μM of various competitor peptides (panels B-M). As shown, only the tri-methyl histone H3 (Lys9) peptide (panel E) competed away binding of the antibody.

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製品番号 サイズ 価格 在庫
95830T		 1 Kit  (4 x 20 µl)

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Product Description

The ATRX/Daxx Antibody Sampler Kit provides an economical means of detecting ATRX and Daxx as well as related histone marks using antibodies. The kit includes enough antibodies to perform two western blot experiments with each primary antibody.

Specificity / Sensitivity

ATRX (D1N2E) Rabbit mAb recognizes endogenous levels of total ATRX protein. Daxx (25C12) Rabbit mAb detects endogenous levels of total Daxx protein. While Daxx has a calculated MW of 81 kDa, it has been shown to run at an apparent MW of 110 kDa at least in part due to posttranslational hyper-phosphorylation (5). Tri-Methyl-Histone H3 (Lys9) (D4W1U) Rabbit mAb detects endogenous levels of histone H3 when tri-methylated on Lys9. This antibody shows some cross-reactivity with histone H3 that is di-methylated on Lys9, but does not cross-react with non-methylated or mono-methylated histone H3 Lys9. This antibody does not detect tri-methyl histone H3 Lys9 when the adjacent Ser10 residue is phosphorylated during mitosis. In addition, this antibody does not cross-react with methylated histone H3 Lys4, Lys27, Lys36, or Lys79. Histone H3 (D1H2) XP® Rabbit mAb detects endogenous levels of total histone H3 protein, including isoforms H3.1, H3.2, and H3.3. This antibody also detects the histone H3 variant CENP-A. This antibody does not cross-react with other core histones.

Source / Purification

ATRX (D1N2E) Rabbit mAb is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Leu1189 of human ATRX protein. Daxx (25C12) Rabbit mAb is produced by immunizing animals with a synthetic peptide corresponding to a region surrounding Gln255 of Daxx protein. Tri-Methyl-Histone H3 (Lys9) (D4W1U) Rabbit mAb is produced by immunizing animals with a synthetic peptide corresponding to residues near the amino terminus of histone H3 in which Lys9 is tri-methylated. Histone H3 (D1H2) XP® Rabbit mAb is produced by immunizing animals with a synthetic peptide corresponding to the carboxy terminus of the human histone H3 protein.

Background

α-thalassemia/mental retardation X-linked (ATRX) is a transcriptional regulator and helicase that belongs to the SNF2 family of chromatin remodeling proteins (1,2). Together with its binding partner death-associated protein 6 (Daxx), ATRX acts as histone chaperone to deposit histone variant H3.3 at repetitive DNA sequences such as telomeric, pericentric, and ribosomal gene repeats (3-6). ATRX is involved in many nuclear functions that ensure proper sister chromatid cohesion during mitosis and chromosome alignment during meiosis (7,8). The ATRX transcriptional regulator also plays a role in the maintenance of telomere integrity and the regulation of gene expression during mammalian development by influencing DNA methylation patterns at high DNA repeat sequences (9,10). Mutations in the corresponding ATRX gene results in ATR-X syndrome, an X-linked disorder characterized by intellectual disabilities, craniofacial abnormalities, and mild α-thalassemia (11,12). Research studies indicate that the loss of ATRX protein occurs in numerous cancers, including pancreatic neuroendocrine tumors (PanNETs) and pediatric glioblastoma, where telomere maintenance occurs independently of telomerase (13-16).

Daxx is a ubiquitously expressed protein that was originally identified through a yeast two-hybrid screen as an interactor with the cytoplasmic domain of Fas. It was found to enhance Fas-mediated apoptosis and activate the JNK pathway (17). However, additional studies have revealed that Daxx is actually a nuclear protein localizing to promyelocytic leukemia oncogenic domains (PODs) (18,19). Nuclear interactions have since been observed with CENP-C (20), Pax3 (22), DNA methyltransferase I (21) and chromatin-associated proteins, including histone deacetylase II, H2A, H2B, H3, H4, and Dek. Roles for Daxx have been suggested in transcriptional repression and cell cycle control. Loss of Daxx in mice leads to embryonic lethality with extensive developmental apoptosis, suggesting a role for Daxx directly or indirectly in suppressing cell death (22). Furthermore, inhibition of Daxx expression using RNAi has confirmed Daxx to be anti-apoptotic and to repress transcriptional activity of targets, including NF-κB and E2F-1 (23).

  1. Clynes, D. et al. (2013) Trends Biochem Sci 38, 461-6.
  2. Picketts, D.J. et al. (1996) Hum Mol Genet 5, 1899-907.
  3. Drané, P. et al. (2010) Genes Dev 24, 1253-65.
  4. Elsässer, S.J. et al. (2012) Nature 491, 560-5.
  5. Lewis, P.W. et al. (2010) Proc Natl Acad Sci U S A 107, 14075-80.
  6. Goldberg, A.D. et al. (2010) Cell 140, 678-91.
  7. Ritchie, K. et al. (2008) J Cell Biol 180, 315-24.
  8. De La Fuente, R. et al. (2004) Dev Biol 272, 1-14.
  9. Wong, L.H. et al. (2010) Genome Res 20, 351-60.
  10. Gibbons, R.J. et al. (2000) Nat Genet 24, 368-71.
  11. Gibbons, R.J. et al. (1995) Cell 80, 837-45.
  12. Gibbons, R.J. et al. (1995) Hum Mol Genet 4 Spec No, 1705-9.
  13. Heaphy, C.M. et al. (2011) Science 333, 425.
  14. Lovejoy, C.A. et al. (2012) PLoS Genet 8, e1002772.
  15. Schwartzentruber, J. et al. (2012) Nature 482, 226-31.
  16. Jiao, Y. et al. (2011) Science 331, 1199-203.
  17. Yang, X. et al. (1997) Cell 89, 1067-76.
  18. Torii, S. et al. (1999) EMBO J 18, 6037-49.
  19. Li, H. et al. (2000) Mol Cell Biol 20, 1784-96.
  20. Pluta, A.F. et al. (1998) J Cell Sci 111 (Pt 14), 2029-41.
  21. Michaelson, J.S. et al. (1999) Genes Dev 13, 1918-23.
  22. Hollenbach, A.D. et al. (1999) EMBO J 18, 3702-11.
  23. Suihko, M.L. and Stackebrandt, E. (2003) J Appl Microbiol 94, 25-34.

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