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26482
Methyl-Histone H3 (Lys4) Antibody Sampler Kit

Methyl-Histone H3 (Lys4) Antibody Sampler Kit #26482

Western Blotting Image 1

Antibody specificity was determined by Western blotting. HeLa and NIH/3T3 cell lysates were probed with Tri-Methyl Histone H3 (Lys4) (C42D8) Rabbit mAb (Panel A) or Tri-Methyl Histone H3 (Lys4) Rabbit mAb pre-adsorbed with 1.5 μM of various competitor peptides (Panels B-M). As shown, only the tri-methyl histone H3 (Lys4) peptide competed away binding of the antibody.

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Chromatin IP-seq Image 2

Chromatin immunoprecipitations were performed with cross-linked chromatin from HCT116 cells and Tri-Methyl-Histone H3 (Lys4) (C42D8) Rabbit mAb, using SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003. DNA Libraries were prepared from 50ng enriched ChIP DNA using NEBNext® Ultra™ II DNA Library Prep Kit for Illumina®, and sequenced on the Illumina NextSeq. The figure shows binding across RPL30, a known target gene of H3K4me3 (see additional figure containing ChIP-qPCR data). 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 Di-Methyl-Histone H3 (Lys4) (C64G9) Rabbit mAb, using SimpleChIP® 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 GAPDH, a known target gene of H3K4me2 (see additional figure containing ChIP-qPCR data). For additional ChIP-seq tracks, please download the product data sheet.

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Western Blotting Image 4

Western blot analysis of whole cell lysates from HeLa, NIH/3T3, C6 and COS cells using Di-Methyl-Histone H3 (Lys4) (C64G9) Rabbit mAb.

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Western Blotting Image 5

Western blot analysis of extracts from various cell lines using Mono-Methyl-Histone H3 (Lys4) (D1A9) XP® Rabbit mAb.

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Chromatin IP-seq Image 6

Chromatin immunoprecipitations were performed with cross-linked chromatin from Hela cells and Mono-Methyl-Histone H3 (Lys4) (D1A9) XP® Rabbit mAb, using SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003. DNA Libraries were prepared from 50ng enriched ChIP DNA using NEBNext® Ultra™ II DNA Library Prep Kit for Illumina®, and sequenced on the Illumina NextSeq. The figure shows binding across ADAM9, a known target gene of H3K4me1 (see additional figure containing ChIP-qPCR data). For additional ChIP-seq tracks, please download the product data sheet.

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Western Blotting Image 7

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

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Western Blotting Image 8

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 9

Western blot analysis of various cell types using Tri-Methyl Histone H3 (Lys4) (C42D8) Rabbit mAb.

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Chromatin IP Image 10

Chromatin immunoprecipitations were performed with cross-linked chromatin from HeLa cells and either Tri-Methyl-Histone H3 (Lys4) (C42D8) 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 RPL30 Exon 3 Primers #7014, SimpleChIP® Human GAPDH Exon 1 Primers #5516, SimpleChIP® Human MyoD1 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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IHC-P (paraffin) Image 11

Immunohistochemical analysis of paraffin-embedded human breast carcinoma using Di-Methyl-Histone H3 (Lys4) (C64G9) Rabbit mAb.

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Chromatin IP Image 12

Chromatin immunoprecipitations were performed with cross-linked chromatin from HeLa cells and either Di-Methyl-Histone H3 (Lys4) (C64G9) 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 RPL30 Exon 3 Primers #7014, SimpleChIP® Human GAPDH Exon 1 Primers #5516, SimpleChIP® Human MyoD1 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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Chromatin IP Image 13

Chromatin immunoprecipitations were performed with cross-linked chromatin from K562 cells and either Mono-Methyl-Histone H3 (Lys4) (D1A9) 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 ADAM9 Intron 11 Primers #73401, human ADAM18 intron 14 primers, human Trio inton 1 primers, and SimpleChIP® Human Trio Exon 57 Primers #90568. 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 14

Flow cytometric analysis of HeLa cells using Mono-Methyl-Histone H3 (Lys4) (D1A9) XP® 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® 647 Conjugate) #4414 was used as a secondary antibody.

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IHC-P (paraffin) Image 15

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

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IHC-P (paraffin) Image 16

Immunohistochemical analysis of paraffin-embedded human colon using Tri-Methyl-Histone H3 (K4) (C42D8) Rabbit mAb in the presence of non-methyl peptide (left) or K4 tri-methyl peptide (right).

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IHC-P (paraffin) Image 17

Immunohistochemical analysis of paraffin-embedded human stomach carcinoma using Di-Methyl-Histone H3 (Lys4) (C64G9) Rabbit mAb.

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IF-IC Image 18

Confocal immunofluorescent analysis of HeLa cells using Mono-Methyl-Histone H3 (Lys4) (D1A9) XP® Rabbit mAb (green) and MEK1/2 (L38C12) Mouse mAb #4694 (blue). Actin filaments were labeled with Dy-554 phalloidin (red).

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Flow Cytometry Image 19

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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Flow Cytometry Image 20

Flow cytometric analysis of human whole blood cells using Tri-Methyl-Histone H3 (Lys4) (C42D8) Rabbit mAb (blue) and 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. Analysis was performed on cells in the lymphocyte gate.

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IF-IC Image 21

Confocal immunofluorescent analysis of HeLa cells using Di-Methyl-Histone H3 (Lys4) (C64G9) Rabbit mAb (green). Actin filaments have been labeled with Alexa Fluor® 555 phalloidin (red).

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IF-IC Image 22

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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IF-IC Image 23

Confocal immunofluorescent analysis of HeLa cells using Tri-Methyl-Histone H3 (Lys4) (C42D8) Rabbit mAb (green). Actin filaments have been labeled with Alexa Fluor® 555 phalloidin (red).

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Product Includes Quantity Applications Reactivity MW(kDa) Isotype
Tri-Methyl-Histone H3 (Lys4) (C42D8) Rabbit mAb 9751 20 µl
  • WB
  • IHC
  • IF
  • F
  • ChIP
H M R Mk Dm Sc 17 Rabbit IgG
Di-Methyl-Histone H3 (Lys4) (C64G9) Rabbit mAb 9725 20 µl
  • WB
  • IP
  • IHC
  • IF
  • F
  • ChIP
H M R Mk 17 Rabbit IgG
Mono-Methyl-Histone H3 (Lys4) (D1A9) XP® Rabbit mAb 5326 20 µl
  • WB
  • IF
  • F
  • 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 (Lys4) Antibody Sampler Kit provides an economical means of detecting levels of mono-, di-, and tri-methyl histone H3 Lys4 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 (Lys4) Antibody Sampler Kit detects endogenous levels of its target protein. Tri-Methyl-Histone H3 (Lys4) (C42D8) Rabbit mAb detects endogenous levels of histone H3 when tri-methylated on Lys4. This antibody shows some cross-reactivity with histone H3 that is di-methylated on Lys4, but does not cross-react with non-methylated or mono-methylated histone H3 Lys4. Di-Methyl-Histone H3 (Lys4) (C64G9) Rabbit mAb detects endogenous levels of histone H3 when di-methylated on Lys4. This antibody shows weak cross-reactivity with histone H3 that is mono-methylated on Lys4 but does not cross-react with non-methylated or tri-methylated histone H3 Lys4. Mono-Methyl-Histone H3 (Lys4) (D1A9) XP® Rabbit mAb detects endogenous levels of histone H3 only when mono-methylated on Lys4. 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 Lys4 antibodies are produced by immunizing rabbits with synthetic peptides corresponding to the amino terminus of histone H3 in which Lys4 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 Lys4 is associated with transcriptional activation. Mono-methyl-histone H3 Lys4 levels are high at trancriptional enhancer elements, with lower levels of mono-methylation found at the promoters of active genes. Tri-methyl-histone H3 Lys4 levels are high at the promoters of active genes, in addition to bivalent, transcriptionally poised genes that also contain the repressive tri-methyl-histone H3 Lys27 modification. Di-methyl-histone H3 Lys4 levels are highest in the 5'-end of transcriptionally active 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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