Revision 1

#76252Store at -20C

1 个试剂盒

(4 x 20 microliters)

Cell Signaling Technology

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For Research Use Only. Not for Use in Diagnostic Procedures.
Product Includes Product # Quantity Mol. Wt Isotype/Source
Tri-Methyl-Histone H3 (Lys9) (D4W1U) Rabbit mAb 13969 20 µl 17 kDa Rabbit IgG
Di-Methyl-Histone H3 (Lys9) (D85B4) XP® Rabbit mAb 4658 20 µl 17 kDa Rabbit IgG
Mono-Methyl-Histone H3 (Lys9) (D1P5R) Rabbit mAb 14186 20 µl 17 kDa Rabbit IgG
Histone H3 (D1H2) XP® Rabbit mAb 4499 20 µl 17 kDa Rabbit IgG
Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl Goat 

Please visit cellsignal.com for individual component applications, species cross-reactivity, dilutions, protocols, and additional product information.

Description

The Methyl-Histone H3 (Lys9) Antibody Sampler Kit provides an economical means of detecting levels of mono-, di-, and tri-methyl histone H3 Lys9 using methyl-specific and control histone H3 antibodies. The kit contains enough primary antibodies to perform at least two western blot experiments.

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 antibodies.

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).

Methylation of histone H3 Lys9 is generally associated with transcriptional repression of constitutive heterochromatin. Tri- and di-methyl-histone H3 Lys9 levels are high in regions of transcriptionally repressed facultative heterochromatin, including transposable elements and centromeric regions. Mono-methyl-histone H3 Lys9 levels are more widely dispersed and found in the bodies of 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.

Background References

    Trademarks and Patents

    Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
    SimpleChIP is a registered trademark of Cell Signaling Technology, Inc.
    XP is a registered trademark of Cell Signaling Technology, Inc.
    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.

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    Revision 1
    #76252

    Methyl-Histone H3 (Lys9) Antibody Sampler Kit

    Methyl-Histone H3 (Lys9) Antibody Sampler Kit: Image 1 Expand Image
    使用 Tri-Methyl-Histone H3 (Lys9) (D4W1U) Rabbit mAb 对不同细胞系的提取物进行蛋白质印迹分析。
    Methyl-Histone H3 (Lys9) Antibody Sampler Kit: Image 2 Expand Image
    使用 Mono-Methyl-Histone H3 (Lys9) (D1P5R) Rabbit mAb 对不同细胞系提取物进行蛋白质印迹分析。
    Methyl-Histone H3 (Lys9) Antibody Sampler Kit: Image 3 Expand Image
    使用 Histone H3 (D1H2) XP® Rabbit mAb 对不同细胞系提取物进行蛋白质印迹分析。
    Methyl-Histone H3 (Lys9) Antibody Sampler Kit: Image 4 Expand Image
    使用 Histone H3 (D1H2) XP® Rabbit mAb 对石蜡包埋的人乳腺癌组织进行免疫组织化学分析。
    Methyl-Histone H3 (Lys9) Antibody Sampler Kit: Image 5 Expand Image
    Western blot analysis of extracts from various cell lines using Di-Methyl-Histone H3 (Lys9) (D85B4) XP® Rabbit mAb.
    Methyl-Histone H3 (Lys9) Antibody Sampler Kit: Image 6 Expand Image
    使用 SimpleChIP® Plus 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 (Lys9) (D85B4) XP® Rabbit mAb 或 Di-Methyl-Histone H3 (Lys9) Antibody #9753 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 XYLT1 基因内的结合作用。如需了解其他 ChIP-seq 情况,请下载产品说明书。
    Methyl-Histone H3 (Lys9) Antibody Sampler Kit: Image 7 Expand Image
    CUT&Tag was performed with HeLa cells and Di-Methyl-Histone H3 (Lys9) (D85B4) XP® 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 the XYLT1 gene.
    Methyl-Histone H3 (Lys9) Antibody Sampler Kit: Image 8 Expand Image
    一抗与靶标蛋白结合之后,与偶联 HRP 的二抗形成复合体。添加 LumiGLO®,在酶催化分解期间发光。
    Methyl-Histone H3 (Lys9) Antibody Sampler Kit: Image 9 Expand Image
    抗体特异性通过蛋白质印迹法确定。对 HeLa 与 NIH/3T3 细胞裂解物使用 Tri-Methyl Histone H3 (Lys9) (D4W1U) Rabbit mAb (小图 A) 或 Tri-Methyl Histone H3 (Lys9) (D4W1U) Rabbit mAb 进行检测,后者使用 1.5 μM 不同竞争肽 (小图 B-M) 作预吸附处理。如图所示,只有三甲基组蛋白 H3 (Lys9) 肽 (小图 E) 与抗体的结合具有优势。
    Methyl-Histone H3 (Lys9) Antibody Sampler Kit: Image 10 Expand Image
    使用 Mono-Methyl-Histone H3 (Lys9) (D1P5R) Rabbit mAb(绿色)对 U-2 OS 细胞进行共聚焦免疫荧光分析。肌动蛋白微丝由 DyLight 554 Phalloidin #13054(红色)标记。
    Methyl-Histone H3 (Lys9) Antibody Sampler Kit: Image 11 Expand Image
    使用 Histone H3 (D1H2) XP® Rabbit mAb 对石蜡包埋的 4T1 同基因乳腺肿瘤组织进行免疫组织化学分析。
    Methyl-Histone H3 (Lys9) Antibody Sampler Kit: Image 12 Expand Image
    Confocal immunofluorescent analysis of HeLa cells using Di-Methyl-Histone H3 (Lys9) (D85B4) XP® Rabbit mAb (green). 肌动蛋白纤丝用 DY-554 Phalloidin 进行标记(红色)。
    Methyl-Histone H3 (Lys9) Antibody Sampler Kit: Image 13 Expand Image
    使用 SimpleChIP® Plus 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 (Lys9) (D85B4) XP® Rabbit mAb 或 Di-Methyl-Histone H3 (Lys9) Antibody #9753 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 16(上图)内的结合,包括 XYLT1 基因(下图)。
    Methyl-Histone H3 (Lys9) Antibody Sampler Kit: Image 14 Expand Image
    CUT&Tag was performed with HeLa cells and Di-Methyl-Histone H3 (Lys9) (D85B4) XP® 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 16 (upper), including the XYLT1 gene (lower).
    Methyl-Histone H3 (Lys9) Antibody Sampler Kit: Image 15 Expand Image
    使用 Tri-Methyl-Histone H3 (Lys9) (D4W1U) Rabbit mAb (绿色) 和 β-Actin (8H10D10) Mouse mAb #3700 (红色),对未经处理(上图)或经 λ 磷酸酶处理的(下图)间期(左图)或有丝分裂期(右图)HeLa 细胞进行共聚焦免疫荧光分析。蓝色伪彩 = DRAQ5® #4084(DNA 荧光染料)。如上所示,当邻近的 Ser10 残基被磷酸化之后,该抗体无法检测有丝分裂细胞中的三甲基组蛋白 H3 Lys9。
    Methyl-Histone H3 (Lys9) Antibody Sampler Kit: Image 16 Expand Image
    使用 Histone H3 (D1H2) XP® Rabbit mAb(绿色)和 β-Tubulin (9F3) Rabbit mAb (Alexa Fluor® 555 Conjugate) #2116(红色)对 HeLa 细胞进行共聚焦免疫荧光分析。
    Methyl-Histone H3 (Lys9) Antibody Sampler Kit: Image 17 Expand Image
    使用 Histone H3 (D1H2) XP® Rabbit mAb 对石蜡包埋的 LL/2 同基因肿瘤组织进行免疫组织化学分析。
    Methyl-Histone H3 (Lys9) Antibody Sampler Kit: Image 18 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 (Lys9) (D85B4) XP® Rabbit mAb 或 Normal Rabbit IgG #2729 后,进行染色质免疫沉淀。使用 SimpleChIP® Human GAPDH Exon 1 Primers #5516、SimpleChIP® Human RPL30 Exon 3 Primers #7014、SimpleChIP® Human AFM Intron 1 Primers #5098和 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。
    Methyl-Histone H3 (Lys9) Antibody Sampler Kit: Image 19 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 (Lys9) (D85B4) XP® Rabbit mAb(实线)对 Jurkat 细胞进行流式细胞分析。Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 用作二抗。
    Methyl-Histone H3 (Lys9) Antibody Sampler Kit: Image 20 Expand Image
    使用以浓度匹配的 Rabbit (DA1E) mAb IgG XP® Isotype Control #3900(虚线)作对照的 Tri-Methyl-Histone H3 (Lys9) (D4W1U) 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® 488 Conjugate) #4412 用作二抗。
    Methyl-Histone H3 (Lys9) Antibody Sampler Kit: Image 21 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 Mono-Methyl-Histone H3 (Lys9) (D1P5R) Rabbit mAb 或 Normal Rabbit IgG #2729 后, 进行染色质免疫沉淀。使用 SimpleChIP® Human GAPDH Promoter Primers #4471、SimpleChIP® Human GAPDH Intron 2 Primers #4478、SimpleChIP® Human MyoD Exon 1 Primers #4490 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。
    Methyl-Histone H3 (Lys9) Antibody Sampler Kit: Image 22 Expand Image
    以浓度匹配的 Rabbit (DA1E) mAb IgG XP® Isotype Control #3900(虚线)作为对照,使用 Histone H3 (D1H2) XP® 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® 488 Conjugate) #4412 作为二抗。
    Methyl-Histone H3 (Lys9) Antibody Sampler Kit: Image 23 Expand Image
    使用 Histone H3 (D1H2) XP® Rabbit mAb 对石蜡包埋的小鼠脑组织进行免疫组织化学分析。
    Methyl-Histone H3 (Lys9) Antibody Sampler Kit: Image 24 Expand Image
    使用 Histone H3 (D1H2) XP® Rabbit mAb 对石蜡包埋的恒河猴肝组织进行免疫组织化学分析。
    Methyl-Histone H3 (Lys9) Antibody Sampler Kit: Image 25 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 Tri-Methyl-Histone H3 (Lys9) (D4W1U) Rabbit mAb 或 Normal Rabbit IgG #2729 进行染色质免疫沉淀。使用 SimpleChIP® Human GAPDH Exon 1 Primers #5516、SimpleChIP® Human AFM Intron 1 Primers #5098、SimpleChIP® Human MYT-1 Exon 1 Primers #4493 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。