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9847
Di-Methyl-Histone H3 Antibody Sampler Kit

Di-Methyl-Histone H3 Antibody Sampler Kit #9847

Chromatin IP-seq Image 1

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 2

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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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

Antibody specificity was determined by Western blotting. HeLa and NIH/3T3 cell extracts were probed with Di-Methyl Histone H3 (Lys36) (C75H12) Rabbit mAb alone (Panel A) or Di-Methyl-Histone H3 (Lys36) (C75H12) Rabbit mAb pre-adsorbed with 1.5 μM of various competitor peptides (Panels B-I). As shown, only the di-methyl-histone H3 (Lys36) peptide competed away binding of the antibody.

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Western Blotting Image 6

Western blot analysis of extracts from various cell lines using Di-Methyl-Histone H3 (Lys27) (D18C8) XP® Rabbit mAb.

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Western Blotting Image 7

Western blot analysis of extracts from HeLa and NIH/3T3 cell lines using Di-Methyl-Histone H3 (Lys79) (D15E8) XP® Rabbit mAb.

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Western Blotting Image 8

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

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Western Blotting Image 9

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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IHC-P (paraffin) Image 10

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

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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 (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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Flow Cytometry Image 12

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 13

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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Western Blotting Image 14

Western blot analysis of extracts from various cell lines using Di-Methyl-Histone H3 (Lys36) (C75H12) Rabbit mAb.

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Flow Cytometry Image 15

Flow cytometric analysis of human peripheral blood lymphocytes using Di-Methyl-Histone H3 (Lys27) (D18C8) 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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Chromatin IP Image 16

Chromatin immunoprecipitations were performed with cross-linked chromatin from HeLa cells and either Di-Methyl-Histone H3 (Lys79) (D15E8) 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 γ-Actin Promoter Primers #5037, SimpleChIP® Human γ-Actin Intron 3 Primers #5047, SimpleChIP® Human GAPDH Promoter Primers #4471, and SimpleChIP® Human GAPDH Intron 2 Primers #4478. 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 17

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

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IHC-P (paraffin) Image 18

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

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IF-IC Image 19

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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IHC-P (paraffin) Image 20

Immunohistochemical analysis of paraffin-embedded human gastric carcinoma using Di-Methyl-Histone H3 (Lys36) (C75H12) Rabbit mAb in the presence of non-methyl peptide (left) or K36 di-methyl peptide (right).

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IF-IC Image 21

Confocal immumunofluorescent analysis of HeLa cells using Di-Methyl-Histone H3 (Lys27) (D18C8) XP® Rabbit mAb (green). Actin filaments have been labled with DY-554 phalloidin (red).

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Flow Cytometry Image 22

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 23

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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Flow Cytometry Image 24

Flow cytometric analysis of HeLa cells using Di-Methyl-Histone H3 (Lys36) (C75H12) 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 25

Chromatin immunoprecipitations were performed with cross-linked chromatin from HeLa cells and either Di-Methyl-Histone H3 (Lys27) (D18C8) 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 MyoD1 Exon 1 Primers #4490, and SimpleChIP® Human AFM Intron 1 Primers #5098. 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.

Learn more about how we get our images
IF-IC Image 26

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 27

Confocal immunofluorescent analysis of HeLa cells using Di-Methyl-Histone H3 (Lys36) (C75H12) Rabbit mAb (green). Actin filaments have been labeled with DY-554 phalloidin (red).

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Product Includes Quantity Applications Reactivity MW(kDa) Isotype
Di-Methyl-Histone H3 (Lys4) (C64G9) Rabbit mAb 9725 20 µl
  • WB
  • IP
  • IHC
  • 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
Di-Methyl-Histone H3 (Lys36) (C75H12) Rabbit mAb 2901 20 µl
  • WB
  • IHC
  • IF
  • F
H M R Mk 17 Rabbit IgG
Di-Methyl-Histone H3 (Lys27) (D18C8) XP® Rabbit mAb 9728 20 µl
  • WB
  • IP
  • IF
  • F
  • ChIP
H M R Mk 17 Rabbit IgG
Di-Methyl-Histone H3 (Lys79) (D15E8) XP® Rabbit mAb 5427 20 µl
  • WB
  • 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 Di-Methyl-Histone H3 Antibody Sampler Kit provides a fast and economical means of evaluating methylation sites on histone H3. The kit contains enough primary and secondary antibodies to perform two western blots.

All antibodies in the Di-Methyl-Histone H3 Antibody Sampler Kit recognize histone H3 only when modified at the indicated site.

Polyclonal antibodies are produced by immunizing animals with synthetic di-methylated peptides corresponding to residues surrounding Lys 4 of human Histone H3. Polyclonal antibodies are purified by protein A and peptide affinity chromatography. Monoclonal antibodies are produced by immunizing animals with synthetic di-methylated peptides corresponding to residues surrounding Lys 9, 27, 36 and 79 of human Histone H3. 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.

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).

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

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
U.S. Patent No. 7,429,487, foreign equivalents, and child patents deriving therefrom.

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