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9783
Tri-Methyl Histone H3 Antibody Sampler Kit
Primary Antibodies
Antibody Sampler Kit

Tri-Methyl Histone H3 Antibody Sampler Kit #9783

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Tri-Methyl Histone H3 Antibody Sampler Kit: Image 1

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 using SimpleChIP® ChIP-seq DNA Library Prep Kit for Illumina® #56795. The figure shows binding across RPL30, a known target gene of H3K4me3 (see additional figure containing ChIP-qPCR data).

Tri-Methyl Histone H3 Antibody Sampler Kit: Image 2

CUT&RUN was performed with HCT 116 cells and Tri-Methyl-Histone H3 (Lys4) (C42D8) Rabbit mAb, using CUT&RUN Assay Kit #86652. DNA Libraries were prepared using SimpleChIP® ChIP-seq DNA Library Prep Kit for Illumina® #56795. The figures show binding across GAPDH, a known target gene of H3K4me3 (see additional figure containing CUT&RUN-qPCR data).

Tri-Methyl Histone H3 Antibody Sampler Kit: Image 3

CUT&RUN was performed with HeLa cells and Tri-Methyl-Histone H3 (Lys27) (C36B11) Rabbit mAb, using CUT&RUN Assay Kit #86652. DNA Libraries were prepared using SimpleChIP® ChIP-seq DNA Library Prep Kit for Illumina® #56795. The figure shows binding across TCEA2 gene.

Tri-Methyl Histone H3 Antibody Sampler Kit: Image 4

Chromatin immunoprecipitations were performed with cross-linked chromatin from Hela cells and either Ezh2 (D2C9) XP® Rabbit mAb #5246 or Tri-Methyl-Histone H3 (Lys27) (C36B11) Rabbit mAb, using SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003. DNA Libraries were prepared using SimpleChIP® ChIP-seq DNA Library Prep Kit for Illumina® #56795. EZH2 and H3K27me3 are known to associate with each other on chromatin. The figure shows binding of both EZH2 and H3K27me3 across MYT1, a known target gene of H3K27me3 (see additional figure containing ChIP-qPCR data).

Tri-Methyl Histone H3 Antibody Sampler Kit: Image 5

Western blot analysis of extracts from various cell lines using Tri-Methyl-Histone H3 (Lys79) Antibody.

Tri-Methyl Histone H3 Antibody Sampler Kit: Image 6

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.

Tri-Methyl Histone H3 Antibody Sampler Kit: 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.

Tri-Methyl Histone H3 Antibody Sampler Kit: Image 8

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 using SimpleChIP® ChIP-seq DNA Library Prep Kit for Illumina® #56795. The figure shows binding across chromosome 8 (upper), including RPL30 (lower), a known target gene of H3K4me3 (see additional figure containing ChIP-qPCR data).

Tri-Methyl Histone H3 Antibody Sampler Kit: Image 9

CUT&RUN was performed with HCT 116 cells and Tri-Methyl-Histone H3 (Lys4) (C42D8) Rabbit mAb, using CUT&RUN Assay Kit #86652. DNA Libraries were prepared using SimpleChIP® ChIP-seq DNA Library Prep Kit for Illumina® #56795. The figures show binding across chromosome 12 (upper), including GAPDH (lower), a known target gene of H3K4me3 (see additional figure containing CUT&RUN-qPCR data).

Tri-Methyl Histone H3 Antibody Sampler Kit: Image 10

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.

Tri-Methyl Histone H3 Antibody Sampler Kit: Image 11

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.

Tri-Methyl Histone H3 Antibody Sampler Kit: Image 12

CUT&RUN was performed with HeLa cells and Tri-Methyl-Histone H3 (Lys27) (C36B11) Rabbit mAb, using CUT&RUN Assay Kit #86652. DNA Libraries were prepared using SimpleChIP® ChIP-seq DNA Library Prep Kit for Illumina® #56795. The figures show binding across chromosome 20 (upper), including TCEA2 gene (lower).

Tri-Methyl Histone H3 Antibody Sampler Kit: Image 13

Chromatin immunoprecipitations were performed with cross-linked chromatin from Hela cells and either Ezh2 (D2C9) XP® Rabbit mAb #5246 or Tri-Methyl-Histone H3 (Lys27) (C36B11) Rabbit mAb, using SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003. DNA Libraries were prepared using SimpleChIP® ChIP-seq DNA Library Prep Kit for Illumina® #56795. EZH2 and H3K27me3 are known to associate with each other on chromatin. The figure shows binding of both EZH2 and H3K27me3 across chromosome 20 (upper), including MYT1 (lower), a known target gene of H3K27me3 (see additional figure containing ChIP-qPCR data).

Tri-Methyl Histone H3 Antibody Sampler Kit: Image 14

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.

Tri-Methyl Histone H3 Antibody Sampler Kit: Image 15

Flow cytometric analysis of HeLa cells using Tri-Methyl-Histone H3 (Lys36) (D5A7) 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® 488 Conjugate) #4412 was used as a secondary antibody.

Tri-Methyl Histone H3 Antibody Sampler Kit: Image 16

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).

Tri-Methyl Histone H3 Antibody Sampler Kit: Image 17

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.

Tri-Methyl Histone H3 Antibody Sampler Kit: Image 18

CUT&RUN was performed with HCT 116 cells and Tri-Methyl-Histone H3 (Lys4) (C42D8) Rabbit mAb or Rabbit (DA1E) mAb IgG XP® Isotype Control (CUT & RUN) #66362, using CUT&RUN Assay Kit #86652. The enriched DNA was quantified by real-time PCR using SimpleChIP® Human GAPDH Exon 1 Primers #5516 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.

Tri-Methyl Histone H3 Antibody Sampler Kit: Image 19

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.

Tri-Methyl Histone H3 Antibody Sampler Kit: Image 20

CUT&RUN was performed with HeLa cells and either Tri-Methyl-Histone H3 (Lys27) (C36B11) Rabbit mAb or Rabbit (DA1E) mAb IgG XP® Isotype Control (CUT&RUN) #66362, using CUT&RUN Assay Kit #86652. The enriched DNA was quantified by real-time PCR using SimpleChIP® Human MYT-1 Exon 1 Primers, SimpleChIP® Human MyoD1 Exon 1 Primers, and SimpleChIP® Human GAPDH Exon 1 Primers. 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.

Tri-Methyl Histone H3 Antibody Sampler Kit: Image 21

Chromatin immunoprecipitations were performed with cross-linked chromatin from HeLa cells and either Tri-Methyl-Histone H3 (Lys27) (C36B11) 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 MyoD1 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.

Tri-Methyl Histone H3 Antibody Sampler Kit: Image 22

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).

Tri-Methyl Histone H3 Antibody Sampler Kit: Image 23

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

Tri-Methyl Histone H3 Antibody Sampler Kit: Image 24

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

Tri-Methyl Histone H3 Antibody Sampler Kit: Image 25

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

Tri-Methyl Histone H3 Antibody Sampler Kit: Image 26

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

Tri-Methyl Histone H3 Antibody Sampler Kit: Image 27

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.

Tri-Methyl Histone H3 Antibody Sampler Kit: Image 28

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

Tri-Methyl Histone H3 Antibody Sampler Kit: Image 29

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.

Tri-Methyl Histone H3 Antibody Sampler Kit: Image 30

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.

Tri-Methyl Histone H3 Antibody Sampler Kit: Image 31

Flow cytometric analysis of human whole blood cells using Tri-Methyl-Histone H3 (Lys27) (C36B11) 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.

Tri-Methyl Histone H3 Antibody Sampler Kit: Image 32

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).

Tri-Methyl Histone H3 Antibody Sampler Kit: Image 33

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).

Tri-Methyl Histone H3 Antibody Sampler Kit: Image 34

Confocal immunofluorescent analysis of HeLa cells using Tri-Methyl-Histone H3 (Lys27) (C36B11) Rabbit mAb (green). Actin filaments have been labeled with DY-554 phalloidin (red).

Tri-Methyl Histone H3 Antibody Sampler Kit: Image 35

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

Tri-Methyl Histone H3 Antibody Sampler Kit: Image 36

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).

Tri-Methyl Histone H3 Antibody Sampler Kit: Image 37

Immunohistochemical analysis of paraffin-embedded human lymphoma using Tri-Methyl-Histone H3 (Lys27) (C36B11) Rabbit mAb in the presence of non-methyl peptide (left) or K27 tri-methyl peptide (right).

Tri-Methyl Histone H3 Antibody Sampler Kit: Image 38

Western blot analysis of extracts from various cell lines using Tri-Methyl-Histone H3 (Lys36) (D5A7) XP® Rabbit mAb.

Tri-Methyl Histone H3 Antibody Sampler Kit: Image 39

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.

Tri-Methyl Histone H3 Antibody Sampler Kit: Image 40

Western blot analysis of various cell lines using Tri-Methyl-Histone H3 (Lys27) (C36B11) Rabbit mAb.

Tri-Methyl Histone H3 Antibody Sampler Kit: Image 41

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

To Purchase # 9783T
Product # Size Price
9783T
1 Kit  (6 x 20 µl) $ 445

Product Includes Quantity Applications Reactivity MW(kDa) Isotype
Tri-Methyl-Histone H3 (Lys4) (C42D8) Rabbit mAb 9751 20 µl
  • WB
  • IHC
  • IF
  • F
  • ChIP
  • C&R
H M R Mk Dm Sc 17 Rabbit IgG
Tri-Methyl-Histone H3 (Lys9) (D4W1U) Rabbit mAb 13969 20 µl
  • WB
  • IP
  • IF
  • F
  • ChIP
H M R Mk 17 Rabbit IgG
Tri-Methyl-Histone H3 (Lys27) (C36B11) Rabbit mAb 9733 20 µl
  • WB
  • IHC
  • IF
  • F
  • ChIP
  • C&R
H M R Mk 17 Rabbit IgG
Tri-Methyl-Histone H3 (Lys36) (D5A7) XP® Rabbit mAb 4909 20 µl
  • WB
  • IHC
  • IF
  • F
  • ChIP
H M R Mk 17 Rabbit IgG
Tri-Methyl-Histone H3 (Lys79) Antibody 4260 20 µl
  • WB
H M R Mk 17 Rabbit 
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 

Product Description

Tri-Methyl Histone H3 Antibody Sampler Kit offers an economical means to evaluate the tri-methylation of Histone H3 on multiple residues. The kit contains enough primary antibody to perform two western blot experiments per primary.

Specificity / Sensitivity

Each modification-state Histone H3 antibody detects endogenous levels of Histone H3 only when tri-methylated on the indicated lysine residue. These antibodies do not cross-react with mono-methylated or di-methylated histone H3, or tri-methylated histone H4 at Lys20. 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. In addition, this antibody does not cross-react with methylated histone H3 Lys4, Lys27, Lys36, or Lys79. Tri-Methyl-Histone H3 (Lys79) Antibody may show slight cross-reactivity toward histone H3 when di-methylated at Lys79, but does not cross-react with histone H3 tri-methylated at Lys4, 9, 27, or 36, or histone H4 at Lys20. Histone H3 (D1H2) XP® Rabbit mAb detects endogenous levels of total histone H3 protein. This antibody does not cross-react with other histones.

Source / Purification

Monoclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to the amino terminus of histone H3 in which Lys4, Lys9, Lys27, and Lys36 are tri-methylated, respectively. Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues surrounding tri-methyl-Lys79. Polyclonal antibodies are purified by protein A and peptide affinity chromatography.

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).

  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.

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