Revision 8
Cell Signaling Technology

Orders: 877-616-CELL (2355) [email protected]

Support: 877-678-TECH (8324)

Web: [email protected] cellsignal.com

3 Trask LaneDanversMassachusetts01923USA
For Research Use Only. Not for Use in Diagnostic Procedures.
Applications:

WB, IP, IHC-P, IF-IC, FC-FP, ChIP, ChIP-seq, C&R, C&T

REACTIVITY:

H M R Mk

SENSITIVITY:

Endogenous

MW (kDa):

17

Source/Isotype:

Rabbit IgG

UniProt ID:

#P68431

Entrez-Gene Id:

8350

Product Information

Product Usage Information

For optimal ChIP and ChIP-seq results, use 10 μl of antibody and 10 μg of chromatin (approximately 4 x 106 cells) per IP. This antibody has been validated using SimpleChIP® Enzymatic Chromatin IP Kits.

The CUT&RUN dilution was determined using CUT&RUN Assay Kit #86652.

The CUT&Tag dilution was determined using CUT&Tag Assay Kit #77552.

Application Dilution
Western Blotting 1:1000
Immunoprecipitation 1:50
Immunohistochemistry (Paraffin) 1:750 - 1:3000
Immunofluorescence (Immunocytochemistry) 1:400 - 1:1600
Flow Cytometry (Fixed/Permeabilized) 1:400 - 1:1600
Chromatin IP 1:50
Chromatin IP-seq 1:50
CUT&RUN 1:50
CUT&Tag 1:50

Storage

Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.

Specificity / Sensitivity

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. In addition, the antibody does not cross-react with methylated histone H3 Lys9, Lys27, Lys36 or histone H4 Lys20.

Species Reactivity:

Human, Mouse, Rat, Monkey

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to the amino terminus of histone H3 in which Lys4 is di-methylated.

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.

Species Reactivity

Species reactivity is determined by testing in at least one approved application (e.g., western blot).

Western Blot Buffer

IMPORTANT: For western blots, incubate membrane with diluted primary antibody in 5% w/v BSA, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.

Applications Key

WB: Western Blotting IP: Immunoprecipitation IHC-P: Immunohistochemistry (Paraffin) IF-IC: Immunofluorescence (Immunocytochemistry) FC-FP: Flow Cytometry (Fixed/Permeabilized) ChIP: Chromatin IP ChIP-seq: Chromatin IP-seq C&R: CUT&RUN C&T: CUT&Tag

Cross-Reactivity Key

H: human M: mouse R: rat Hm: hamster Mk: monkey Vir: virus Mi: mink C: chicken Dm: D. melanogaster X: Xenopus Z: zebrafish B: bovine Dg: dog Pg: pig Sc: S. cerevisiae Ce: C. elegans Hr: horse GP: Guinea Pig Rab: rabbit All: all species expected

Trademarks and Patents

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
Alexa Fluor is a registered trademark of Life Technologies Corporation.
U.S. Patent No. 7,429,487, foreign equivalents, and child patents deriving therefrom.
All other trademarks are the property of their respective owners. Visit cellsignal.com/trademarks for more information.

限制使用

除非 CST 的合法授书代表以书面形式书行明确同意,否书以下条款适用于 CST、其关书方或分书商提供的书品。 任何书充本条款或与本条款不同的客书条款和条件,除非书 CST 的合法授书代表以书面形式书独接受, 否书均被拒书,并且无效。

专品专有“专供研究使用”的专专或专似的专专声明, 且未专得美国食品和专品管理局或其他外国或国内专管机专专专任何用途的批准、准专或专可。客专不得将任何专品用于任何专断或治专目的, 或以任何不符合专专声明的方式使用专品。CST 专售或专可的专品提供专作专最专用专的客专,且专用于研专用途。将专品用于专断、专防或治专目的, 或专专售(专独或作专专成)或其他商专目的而专专专品,均需要 CST 的专独专可。客专:(a) 不得专独或与其他材料专合向任何第三方出售、专可、 出借、捐专或以其他方式专专或提供任何专品,或使用专品制造任何商专专品,(b) 不得复制、修改、逆向工程、反专专、 反专专专品或以其他方式专专专专专品的基专专专或技专,或使用专品开专任何与 CST 的专品或服专专争的专品或服专, (c) 不得更改或专除专品上的任何商专、商品名称、徽专、专利或版专声明或专专,(d) 只能根据 CST 的专品专售条款和任何适用文档使用专品, (e) 专遵守客专与专品一起使用的任何第三方专品或服专的任何专可、服专条款或专似专专

Revision 8
#9725

Di-Methyl-Histone H3 (Lys4) (C64G9) Rabbit mAb

Western Blotting Image 1: Di-Methyl-Histone H3 (Lys4) (C64G9) Rabbit mAb Expand Image
使用 Di-Methyl-Histone H3 (Lys4) (C64G9) Rabbit mAb,对 HeLa、NIH/3T3、C6 和 COS 细胞的全细胞裂解物进行蛋白质印迹分析。
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Immunohistochemistry Image 1: Di-Methyl-Histone H3 (Lys4) (C64G9) Rabbit mAb Expand Image
使用 Di-Methyl-Histone H3 (Lys4) (C64G9) Rabbit mAb,对石蜡包埋的人类乳腺癌进行免疫组织化学分析。
Immunohistochemistry Image 2: Di-Methyl-Histone H3 (Lys4) (C64G9) Rabbit mAb Expand Image
使用 Di-Methyl-Histone H3 (Lys4) (C64G9) Rabbit mAb,对石蜡包埋的人类胃癌进行免疫组织化学分析。
Immunofluorescence Image 1: Di-Methyl-Histone H3 (Lys4) (C64G9) Rabbit mAb Expand Image
使用 Di-Methyl-Histone H3 (Lys4) (C64G9) Rabbit mAb (绿色),对 HeLa 细胞进行共聚焦免疫荧光分析。肌动蛋白纤丝用 Alexa Fluor®555 phalloidin(红色)进行标记。
Flow Cytometry Image 1: Di-Methyl-Histone H3 (Lys4) (C64G9) Rabbit mAb Expand Image
以浓度匹配的 Rabbit (DA1E) mAb IgG XP® Isotype Control #3900(虚线)作为对照,使用 Di-Methyl-Histone H3 (Lys4) (C64G9) Rabbit mAb(实线)对 HeLa 细胞进行流式细胞分析。Anti-rabbit IgG (H+L)、F(ab')2 Fragment (Alexa Fluor® 647 Conjugate) #4414 作为二抗。
Chromatin Immunoprecipitation Image 1: Di-Methyl-Histone H3 (Lys4) (C64G9) Rabbit mAb Expand Image
使用 SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9005,对 HeLa 细胞中提取的交联染色质,在加入 Di-Methyl-Histone H3 (Lys4) (C64G9) Rabbit mAb 后,进行染色质免疫沉淀。使用 SimpleChIP® ChIP-seq DNA Library Prep Kit for Illumina® #56795 制成 DNA 库。本图显示整个 GAPDH (H3K4me2 的一个已知靶标基因)范围内的结合作用(参见包含 ChIP-qPCR 数据的其他结果图)。如需了解其他 ChIP-seq 情况,请下载产品说明书。
Chromatin Immunoprecipitation Image 2: Di-Methyl-Histone H3 (Lys4) (C64G9) Rabbit mAb Expand Image
使用 SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9005,对 HeLa 细胞中提取的交联染色质,在加入 Di-Methyl-Histone H3 (Lys4) (C64G9) Rabbit mAb 后,进行染色质免疫沉淀。使用 SimpleChIP® ChIP-seq DNA Library Prep Kit for Illumina® #56795 制成 DNA 库。结果图显示在染色体12(上图)内的结合,包括 H3K4me2 的已知靶标基因 GAPDH(下图)(参见包含 ChIP-qPCR 数据的其他结果图)。
Chromatin Immunoprecipitation Image 3: Di-Methyl-Histone H3 (Lys4) (C64G9) Rabbit mAb Expand Image
使用 SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003,对 HeLa 细胞中提取的交联染色质,在加入 Di-Methyl-Histone H3 (Lys4) (C64G9) Rabbit mAb 或 Normal Rabbit IgG #2729 后,进行染色质免疫沉淀。使用 SimpleChIP® Human RPL30 Exon 3 Primers #7014、SimpleChIP® Human GAPDH Exon 1 Primers #5516、SimpleChIP® Human MyoD1 Exon 1 Primers #4490 以及 SimpleChIP® Human α Satellite Repeat Primers #4486,通过实时 PCR 对富集的 DNA 进行量化。将每份样品中免疫沉淀的 DNA 的量表现为相对于所输入染色质总量(等于 1)的信号。
CUT and RUN Image 1: Di-Methyl-Histone H3 (Lys4) (C64G9) Rabbit mAb Expand Image
使用 CUT&RUN Assay Kit #86652 对 Hela 细胞和 Di-Methyl-Histone H3 (Lys4) (C64G9) Rabbit mAb 或 Mono-Methyl-Histone H3 (Lys36) (D9J1D) Rabbit mAb #14111 进行 CUT&RUN 检测。使用 DNA Library Prep Kit for Illumina Systems (ChIP-seq, CUT&RUN) #56795 制备 DNA 库。该图显示结合作用遍及 HOXA11 基因。
CUT and RUN Image 2: Di-Methyl-Histone H3 (Lys4) (C64G9) Rabbit mAb Expand Image
使用 CUT&RUN Assay Kit #86652 对 Hela 细胞和 Di-Methyl-Histone H3 (Lys4) (C64G9) Rabbit mAb 或 Mono-Methyl-Histone H3 (Lys36) (D9J1D) Rabbit mAb #14111 进行 CUT&RUN 检测。使用 DNA Library Prep Kit for Illumina Systems (ChIP-seq, CUT&RUN) #56795 制备 DNA 库。这些图显示结合作用遍及 HOXA 基因簇(上图)和 HOXD(下图)基因簇。
CUT and RUN Image 3: Di-Methyl-Histone H3 (Lys4) (C64G9) Rabbit mAb Expand Image
使用 CUT&RUN Assay Kit # 86652 对 Hela 细胞和 Di-Methyl-Histone H3 (Lys4) (C64G9) Rabbit mAb 或 Rabbit (DA1E) mAb IgG XP® Isotype Control (CUT&RUN) #66362 进行 CUT&RUN 检测。使用 SimpleChIP® Human HoxA1 Intron 1 Primers #7707和 SimpleChIP® Human α Satellite Repeat Primers #4486,通过实时 PCR 对富集的 DNA 进行定量分析。将每份样品中免疫沉淀的 DNA 的量表现为相对于所输入染色质总量(等于 1)的信号。
CUT and Tag Image 1: Di-Methyl-Histone H3 (Lys4) (C64G9) Rabbit mAb Expand Image
CUT&Tag was performed with HeLa cells and Di-Methyl-Histone H3 (Lys4) (C64G9) Rabbit mAb, using CUT&Tag Assay Kit #77552. DNA library was prepared using CUT&Tag Dual Index Primers and PCR Master Mix for Illumina Systems #47415. The figure shows binding across GAPDH, a known target gene of H3K4me2 (see our ChIP-qPCR figure).
CUT and Tag Image 2: Di-Methyl-Histone H3 (Lys4) (C64G9) Rabbit mAb Expand Image
CUT&Tag was performed with HeLa cells and Di-Methyl-Histone H3 (Lys4) (C64G9) Rabbit mAb, using CUT&Tag Assay Kit #77552. DNA library was prepared using CUT&Tag Dual Index Primers and PCR Master Mix for Illumina Systems #47415. The figures show binding across chromosome 12 (upper), including GAPDH (lower), a known target gene of H3K4me2 (see our ChIP-qPCR figure).