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76252
Methyl-Histone H3 (Lys9) Antibody Sampler Kit

Methyl-Histone H3 (Lys9) Antibody Sampler Kit #76252

Western Blotting Image 1

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

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Chromatin IP-seq Image 2

Chromatin immunoprecipitations were performed with cross-linked chromatin from Hela cells and either HP1β (D2F2) XP® Rabbit mAb #8676 or Tri-Methyl-Histone H3 (Lys9) (D4W1U) Rabbit mAb, using SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003. DNA Libraries were prepared from 5ng enriched ChIP DNA for HP1β ChIP-seq and 50ng enriched ChIP DNA for H3K9me3 ChIP-seq using NEBNext® Ultra™ II DNA Library Prep Kit for Illumina®, and sequenced on the Illumina NextSeq. HP1β and H3K9me3 are known to associate with each other on chromatin. The figure shows binding of both HP1β and H3K9me3 across ZNF genes on chromosome 19, known target genes of both HP1β and H3K9me3. For additional ChIP-seq tracks, please download the product data sheet.

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Chromatin IP-seq Image 3

Chromatin immunoprecipitations were performed with cross-linked chromatin from HeLa cells and either Di-Methyl-Histone H3 (Lys9) (D85B4) XP® Rabbit mAb or Di-Methyl-Histone H3 (Lys9) Antibody #9753, using SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. DNA Libraries were prepared from 50 ng enriched ChIP DNA using NEBNext® Ultra™ II DNA Library Prep Kit for Illumina®, and sequenced on the Illumina NextSeq. The figure shows binding across XYLT1 gene. For additional ChIP-seq tracks, please download the product data sheet.

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Western Blotting Image 4

Western blot analysis of extracts from various cell lines using Di-Methyl-Histone H3 (Lys9) (D85B4) XP Rabbit mAb.

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Western Blotting Image 5

Western blot analysis of extracts from various cell lines using Mono-Methyl-Histone H3 (Lys9) (D1P5R) Rabbit mAb.

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Western Blotting Image 6

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

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Western Blotting Image 7

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.

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Western Blotting Image 8

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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Chromatin IP Image 9

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.

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Flow Cytometry Image 10

Flow cytometric analysis of HeLa cells using Di-Methyl-Histone H3 (Lys9) (D85B4) XP Rabbit mAb (blue) compared to concentration-matched Rabbit (DA1E) mAb IgG XP Isotype Control #3900 (red). Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor 488 Conjugate) #4412 was used as a secondary antibody.

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Chromatin IP Image 11

Chromatin immunoprecipitations were performed with cross-linked chromatin from HeLa cells and either Di-Methyl-Histone H3 (Lys9) (D85B4) XP® 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 RPL30 Exon 3 Primers #7014, SimpleChIP® Human AFM Intron 1 Primers #5098, 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.

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IF-IC Image 12

Confocal immunofluorescent analysis of U-2 OS cells using Mono-Methyl-Histone H3 (Lys9) (D1P5R) Rabbit mAb (green). Actin filaments were labeled with DyLight™ 554 Phalloidin #13054 (red).

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IHC-P (paraffin) Image 13

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

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Flow Cytometry Image 14

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

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IF-IC Image 15

Confocal immunofluorescent analysis of HeLa cells using Di-Methyl-Histone H3 (Lys9) (D85B4) XP Rabbit mAb

(green). Actin filaments have been labeled with DY-554 phalloidin (red).

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Chromatin IP Image 16

Chromatin immunoprecipitations were performed with cross-linked chromatin from HeLa cells and either Mono-Methyl-Histone H3 (Lys9) (D1P5R) 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 Promoter Primers #4471, SimpleChIP® Human GAPDH Intron 2 Primers #4478, SimpleChIP® Human MyoD Exon 1 Primers #4490, 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.

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Flow Cytometry Image 17

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.

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IF-IC Image 18

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.

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IF-IC Image 19

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).

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Product Includes Quantity Applications Reactivity MW(kDa) Isotype
Tri-Methyl-Histone H3 (Lys9) (D4W1U) Rabbit mAb 13969 20 µl
  • WB
  • IP
  • IF
  • F
  • ChIP
H M R Mk 17 Rabbit IgG
Di-Methyl-Histone H3 (Lys9) (D85B4) XP® Rabbit mAb 4658 20 µl
  • WB
  • IP
  • IF
  • F
  • ChIP
H M R Mk 17 Rabbit IgG
Mono-Methyl-Histone H3 (Lys9) (D1P5R) Rabbit mAb 14186 20 µl
  • WB
  • IP
  • IF
  • ChIP
H M R Mk 17 Rabbit IgG
Histone H3 (D1H2) XP® Rabbit mAb 4499 20 µl
  • WB
  • IHC
  • IF
  • F
H M R Mk 17 Rabbit IgG
Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl
  • WB
Goat 

The Methyl-Histone H3 (Lys9) Antibody Sampler Kit provides an economical means of detecting levels of mono-, di-, and tri-methyl histone H3 Lys9 using methyl-specific and control histone H3 antibodies. The kit contains enough primary antibodies to perform at least two western blot experiments.

Each antibody in the Methyl-Histone H3 (Lys9) Antibody Sampler Kit detects endogenous levels of its target protein. 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. Di-Methyl-Histone H3 (Lys9) (D85B4) XP® Rabbit mAb detects endogenous levels of histone H3 only when di-methylated on Lys9. The antibody does not cross-react with non-methylated, mono-methylated or tri-methylated Histone H3 Lys9. Mono-Methyl-Histone H3 (Lys9) (D1P5R) Rabbit mAb recognizes endogenous levels of histone H3 protein only when mono-methylated at Lys9. This antibody does not cross-react with non-methylated, di-methylated, or tri-methylated Lys9. Histone H3 (D1H2) XP® Rabbit mAb detects endogenous levels of total Histone H3 protein, including isoforms H3.1, H3.2, H3.3, and the variant histone CENP-A. This antibody does not cross-react with other core histones.

Monoclonal methyl-histone H3 Lys9 antibodies are produced by immunizing rabbits with synthetic peptides corresponding to the amino terminus of histone H3 in which Lys9 is mono-, di-, or tri-methylated. The control histone H3 monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to the carboxy terminus of the human histone H3 protein.

The nucleosome, made up of four core histone proteins (H2A, H2B, H3, and H4), is the primary building block of chromatin. Originally thought to function as a static scaffold for DNA packaging, histones have now been shown to be dynamic proteins, undergoing multiple types of post-translational modifications, including acetylation, phosphorylation, methylation, and ubiquitination (1). Histone methylation is a major determinant for the formation of active and inactive regions of the genome and is crucial for the proper programming of the genome during development (2,3). Arginine methylation of histones H3 (Arg2, 17, 26) and H4 (Arg3) promotes transcriptional activation and is mediated by a family of protein arginine methyltransferases (PRMTs), including the co-activators PRMT1 and CARM1 (PRMT4) (4). In contrast, a more diverse set of histone lysine methyltransferases has been identified, all but one of which contain a conserved catalytic SET domain originally identified in the Drosophila Su(var)3-9, Enhancer of zeste, and Trithorax proteins. Lysine methylation occurs primarily on histones H3 (Lys4, 9, 27, 36, 79) and H4 (Lys20) and has been implicated in both transcriptional activation and silencing (4). Methylation of these lysine residues coordinates the recruitment of chromatin modifying enzymes containing methyl-lysine binding modules such as chromodomains (HP1, PRC1), PHD fingers (BPTF, ING2), tudor domains (53BP1), and WD-40 domains (WDR5) (5-8). The discovery of histone demethylases such as PADI4, LSD1, JMJD1, JMJD2, and JHDM1 has shown that methylation is a reversible epigenetic marker (9).

Methylation of histone H3 Lys9 is generally associated with transcriptional repression of constitutive heterochromatin. Tri- and di-methyl-histone H3 Lys9 levels are high in regions of transcriptionally repressed facultative heterochromatin, including transposable elements and centromeric regions. Mono-methyl-histone H3 Lys9 levels are more widely dispersed and found in the bodies of active and inactive genes.

  1. Peterson, C.L. and Laniel, M.A. (2004) Curr Biol 14, R546-51.
  2. Kubicek, S. et al. (2006) Ernst Schering Res Found Workshop , 1-27.
  3. Lin, W. and Dent, S.Y. (2006) Curr Opin Genet Dev 16, 137-42.
  4. Lee, D.Y. et al. (2005) Endocr Rev 26, 147-70.
  5. Daniel, J.A. et al. (2005) Cell Cycle 4, 919-26.
  6. Shi, X. et al. (2006) Nature 442, 96-9.
  7. Wysocka, J. et al. (2006) Nature 442, 86-90.
  8. Wysocka, J. et al. (2005) Cell 121, 859-72.
  9. Trojer, P. and Reinberg, D. (2006) Cell 125, 213-7.
Entrez-Gene Id
8350
Swiss-Prot Acc.
P68431
For Research Use Only. Not For Use In Diagnostic Procedures.

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U.S. Patent No. 7,429,487, foreign equivalents, and child patents deriving therefrom.
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