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46599
Methyl-Histone H3 (Lys36) Antibody Sampler Kit

Methyl-Histone H3 (Lys36) Antibody Sampler Kit #46599

Chromatin IP-seq Image 1

Chromatin immunoprecipitations were performed with cross-linked chromatin from HeLa cells and Tri-Methyl-Histone H3 (Lys36) (D5A7) 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 ACTG1/γ-Actin, a known target gene of H3K36me3 (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 extracts from various cell lines using Tri-Methyl-Histone H3 (Lys36) (D5A7) XP® Rabbit mAb.

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Western Blotting Image 3

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 4

Western blot analysis of extracts from various cell lines using Mono-Methyl-Histone H3 (Lys36) (D9J1D) Rabbit mAb.

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Western Blotting Image 5

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

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Western Blotting Image 6

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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Chromatin IP Image 7

Chromatin immunoprecipitations were performed with cross-linked chromatin from HeLa cells and either Tri-Methyl-Histone H3 (Lys36) (D5A7) 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 8

Immunohistochemical analysis of paraffin-embedded human papillary carcinoma of the breast using Tri-Methyl-Histone H3(K36) (D5A7) XP(R) Rabbit mAb.

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Western Blotting Image 9

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

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Western Blotting Image 10

Antibody specificity was determined by western blotting. HeLa and NIH/3T3 cell extracts were probed with Mono-Methyl-Histone H3 (Lys36) (D9J1D) Rabbit mAb alone (A) or Mono-Methyl-Histone H3 (Lys36) (D9J1D) Rabbit mAb

pre-adsorbed with 1.5 μM of various competitor peptides (B-I). As shown, only the mono-methyl-histone H3 (Lys36)

peptide competed away binding of the antibody (C).

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IHC-P (paraffin) Image 11

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

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IHC-P (paraffin) Image 12

Immunohistochemical analysis of paraffin-embedded human colon carcinoma using Tri-Methyl-Histone H3(K36) (D5A7) XP(R) Rabbit mAb.

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IHC-P (paraffin) Image 13

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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Flow Cytometry Image 14

Flow cytometric analysis of HeLa cells using Mono-Methyl-Histone H3 (Lys36) (D9J1D) 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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Flow Cytometry Image 15

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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IHC-P (paraffin) Image 16

Immunohistochemical analysis of paraffin-embedded 786-O cell pellet (left, positive) or A498 cell pellet (right, negative) using Tri-Methyl-Histone H3(K36) (D5A7) XP(R) Rabbit mAb. Note that the A498 cell line harbors a SETD2 mutation.

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Flow Cytometry Image 17

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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IF-IC Image 18

Confocal immunofluorescent analysis of HeLa cells using Mono-Methyl-Histone H3 (Lys36) (D9J1D) Rabbit mAb (green) and β-Actin (8H10D10) Mouse mAb #3700 (red).

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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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IHC-P (paraffin) Image 20

Immunohistochemical analysis of paraffin-embedded human serous papillary carcinoma of the ovary using Tri-Methyl-Histone H3(K36) (D5A7) XP(R) Rabbit mAb in the presence of non-methyl peptide (left) or K36 tri-methyl peptide (right).

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IF-IC Image 21

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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Chromatin IP Image 22

Chromatin immunoprecipitations were performed with cross-linked chromatin from HeLa cells and either Mono-Methyl-Histone H3 (Lys36) (D9J1D) 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 AFM Intron 1 Primers #5098, SimpleChIP® Human GAPDH Exon 1 Primers #5516, SimpleChIP® Human MyoD Exon 1 Primers #4490, and SimpleChIP® Human MYT-1 Exon 1 Primers #4493. 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 23

Immunohistochemical analysis of paraffin-embedded human prostate carcinoma using Tri-Methyl-Histone H3(K36) (D5A7) XP(R) Rabbit mAb.

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Flow Cytometry Image 24

Flow cytometric analysis of HeLa cells using Tri-Methyl-Histone H3 (Lys36) (D5A7) 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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IF-IC Image 25

Confocal immunofluorescent analysis of HeLa cells using Tri-Methyl-Histone H3 (Lys36) (D5A7) XP® Rabbit mAb (green) and COX IV (4D11-B3-E8) Mouse mAb #11967 (red).

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Product Includes Quantity Applications Reactivity MW(kDa) Isotype
Tri-Methyl-Histone H3 (Lys36) (D5A7) XP® Rabbit mAb 4909 20 µl
  • WB
  • IHC
  • 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
Mono-Methyl-Histone H3 (Lys36) (D9J1D) Rabbit mAb 14111 20 µl
  • WB
  • IP
  • 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 (Lys36) Antibody Sampler Kit provides an economical means of detecting levels of mono-, di-, and tri-methyl histone H3 Lys36 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 (Lys36) Antibody Sampler Kit detects endogenous levels of its target protein. Tri-Methyl-Histone H3 (Lys36) (D5A7) XP® Rabbit mAb detects endogenous levels of histone H3 only when tri-methylated on Lys36. Di-Methyl-Histone H3 (Lys36) (C75H12) Rabbit mAb detects endogenous levels of histone H3, only when di-methylated on Lys36. Mono-Methyl-Histone H3 (Lys36) (D9J1D) Rabbit mAb recognizes endogenous levels of histone H3 only when mono-methylated at Lys36. 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 Lys36 antibodies are produced by immunizing rabbits with synthetic peptides corresponding to the amino terminus of histone H3 in which Lys36 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 Lys36 is associated with transcriptionally active genes. Tri- and di-methyl-histone H3 Lys36 levels are high in the bodies of active genes, where these marks function to repress intragenic transcription initiation and regulate mRNA splicing. Mono-methyl-histone H3 Lys36 levels are high in the bodies of both 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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