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76252
Methyl-Histone H3 (Lys9) Antibody Sampler Kit
Primary Antibodies
Antibody Sampler Kit

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

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Methyl-Histone H3 (Lys9) Antibody Sampler Kit: Image 1

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.

Methyl-Histone H3 (Lys9) Antibody Sampler Kit: Image 2

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.

Methyl-Histone H3 (Lys9) Antibody Sampler Kit: Image 3

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.

Methyl-Histone H3 (Lys9) Antibody Sampler Kit: Image 4

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.

Methyl-Histone H3 (Lys9) Antibody Sampler Kit: Image 5

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.

Methyl-Histone H3 (Lys9) Antibody Sampler Kit: Image 6

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.

Methyl-Histone H3 (Lys9) Antibody Sampler Kit: Image 7

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.

Methyl-Histone H3 (Lys9) Antibody Sampler Kit: Image 8

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).

Methyl-Histone H3 (Lys9) Antibody Sampler Kit: Image 9

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.

Methyl-Histone H3 (Lys9) Antibody Sampler Kit: Image 10

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).

Methyl-Histone H3 (Lys9) Antibody Sampler Kit: Image 11

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).

Methyl-Histone H3 (Lys9) Antibody Sampler Kit: Image 12

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

Methyl-Histone H3 (Lys9) Antibody Sampler Kit: Image 13

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

Methyl-Histone H3 (Lys9) Antibody Sampler Kit: Image 14

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

Methyl-Histone H3 (Lys9) Antibody Sampler Kit: Image 15

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

Methyl-Histone H3 (Lys9) Antibody Sampler Kit: Image 16

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

Methyl-Histone H3 (Lys9) Antibody Sampler Kit: Image 17

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.

To Purchase # 76252T
Product # Size Price
76252T
1 Kit  (4 x 20 µl) $ 327

Product Description

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.

Specificity / Sensitivity

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.

Source / Purification

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.

Background

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, 859-872.
  9. Trojer, P. and Reinberg, D. (2006) Cell 125, 213-7.

Pathways & Proteins

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For Research Use Only. Not For Use In Diagnostic Procedures.
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