Revision 4
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:

IF-IC, DB

REACTIVITY:

All

SENSITIVITY:

Endogenous

MW (kDa):

Source/Isotype:

Mouse IgG1

Product Information

Product Usage Information

Application Dilution
Immunofluorescence (Immunocytochemistry) 1:400
DNA Dot Blot 1:1000

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

5-Hydroxymethylcytosine (5-hmC) (HMC31) Mouse mAb recognizes endogenous levels of 5-hmC; however many cells and tissues contain very low levels of 5-hmC that may fall below the detection limits of this antibody. This antibody has been validated using ELISA, dot blot, and MeDIP assays and shows high specificity for 5-hmC.

Species Reactivity:

All Species Expected

Source / Purification

Monoclonal antibody is produced by immunizing animals with 5-hydroxymethylcytidine.

Background

Methylation of DNA at cytosine residues is a heritable, epigenetic modification that is critical for proper regulation of gene expression, genomic imprinting, and mammalian development (1,2). 5-methylcytosine is a repressive epigenetic mark established de novo by two enzymes, DNMT3a and DNMT3b, and is maintained by DNMT1 (3, 4). 5-methylcytosine was originally thought to be passively depleted during DNA replication. However, subsequent studies have shown that Ten-Eleven Translocation (TET) proteins TET1, TET2, and TET3 can catalyze the oxidation of methylated cytosine to 5-hydroxymethylcytosine (5-hmC) (5). Additionally, TET proteins can further oxidize 5-hmC to form 5-formylcytosine (5-fC) and 5-carboxylcytosine (5-caC), both of which are excised by thymine-DNA glycosylase (TDG), effectively linking cytosine oxidation to the base excision repair pathway and supporting active cytosine demethylation (6,7).
TET protein-mediated cytosine hydroxymethylation was initially demonstrated in mouse brain and embryonic stem cells (5, 8). Since then this modification has been discovered in many tissues, with the highest levels found in the brain (9). While 5-fC and 5-caC appear to be short-lived intermediate species, there is mounting evidence showing that 5-hmC is a distinct epigenetic mark with various unique functions (10,11). The modified base itself is stable in vivo and interacts with various readers including MeCP2 (11,12). The global level of 5-hmC increases during brain development and 5-hmC is enriched at promoter regions and poised enhancers. Furthermore, there is an inverse correlation between levels of 5-hmC and histone H3K9 and H3K27 trimethylation, suggesting a role for 5-hmC in gene activation (12). Lower amounts of 5-hmC have been reported in various cancers including myeloid leukemia and melanoma (13,14).

  1. Hermann, A. et al. (2004) Cell Mol Life Sci 61, 2571-87.
  2. Turek-Plewa, J. and Jagodziński, P.P. (2005) Cell Mol Biol Lett 10, 631-47.
  3. Okano, M. et al. (1999) Cell 99, 247-57.
  4. Li, E. et al. (1992) Cell 69, 915-26.
  5. Tahiliani, M. et al. (2009) Science 324, 930-5.
  6. He, Y.F. et al. (2011) Science 333, 1303-7.
  7. Ito, S. et al. (2011) Science 333, 1300-3.
  8. Kriaucionis, S. and Heintz, N. (2009) Science 324, 929-30.
  9. Globisch, D. et al. (2010) PLoS One 5, e15367.
  10. Gao, Y. et al. (2013) Cell Stem Cell 12, 453-69.
  11. Mellén, M. et al. (2012) Cell 151, 1417-30.
  12. Wen, L. et al. (2014) Genome Biol 15, R49.
  13. Delhommeau, F. et al. (2009) N Engl J Med 360, 2289-301.
  14. Lian, C.G. et al. (2012) Cell 150, 1135-46.

Species Reactivity

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

Applications Key

IF-IC: Immunofluorescence (Immunocytochemistry) DB: DNA Dot Blot

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.
SimpleChIP is a registered trademark of Cell Signaling Technology, Inc.
XP is a registered trademark of Cell Signaling Technology, Inc.
All other trademarks are the property of their respective owners. Visit cellsignal.com/trademarks for more information.

限制使用

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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 4
#51660

5-Hydroxymethylcytosine (5-hmC) (HMC31) Mouse mAb

Immunofluorescence Image 1: 5-Hydroxymethylcytosine (5-hmC) (HMC31) Mouse mAb Expand Image
使用 5-Hydroxymethylcytosine (5-hmC) (HMC31) Mouse mAb(绿色)和 DYKDDDDK Tag Antibody #2368(红色),对转染表达带有 DDK 标签的 TET1 催化结构域 (TET1-CD) 载体的 293T 细胞进行共聚焦免疫荧光分析。蓝色伪彩 = DRAQ5® #4084(DNA 荧光染料)。正如预期的一样,表达 TET1-CD(红色)的 293T 细胞出现 5-羟甲基胞嘧啶(绿色)水平升高。
Dot Blot Image 1: 5-Hydroxymethylcytosine (5-hmC) (HMC31) Mouse mAb Expand Image
5-Hydroxymethylcytosine (5-hmC) (HMC31) Mouse mAb 的特异性通过斑点印迹实验进行确定。专门使用未修饰的胞嘧啶、5-甲基胞嘧啶 (5-mC)、5-羟甲基胞嘧啶(5-hmC)、5-羧基胞嘧啶 (5-caC) 或 5-甲酰基胞嘧啶 (5-fC),通过 PCR 产生一个 387 碱基对 DNA 片段的相同序列。单独 DNA 片段被转移到尼龙膜上成为印迹,经紫外线交联并使用 5-Hydroxymethylcytosine (5-hmC) (HMC31) Mouse mAb 进行检测。上小图显示抗体仅结合含 5-hmC 的 DNA 片段,而下小图则显示用亚甲蓝染色的膜。
Product Image 1: 5-Hydroxymethylcytosine (5-hmC) (HMC31) Mouse mAb Expand Image
5-Hydroxymethylcytosine (5-hmC) (HMC31) Mouse mAb 的特异性通过 ELISA 进行确定。使用含有未修饰的胞嘧啶或差异性修饰的胞嘧啶(5-mC、5-hmC、5-caC、5-fC)的单链 DNA 寡聚物滴定抗体。如图表所示,该抗体仅结合含 5-hmC 的寡核苷酸。
Product Image 2: 5-Hydroxymethylcytosine (5-hmC) (HMC31) Mouse mAb Expand Image
使用 1 μg 小鼠胚胎干细胞基因组 DNA 与 10 μl 5-Hydroxymethylcytosine (5-hmC) (HMC31) Mouse mAb 或 10 μl Mouse (G3A1) mAb IgG1 Isotype Control (DIP Formulated) # 进行 DNA 免疫沉淀。使用 mouse Aqp2 exon 1 primers, SimpleDIP Mouse Intracisternal-A Particle (IAP) LTR Primers、mouse Lamc3 exon 1 primers 和 SimpleChIP® Mouse GAPDH Intron 2 Primers #8986,通过实时 PCR 对富集的 DNA 进行定量分析。每种样品中免疫沉淀的 DNA 的量以相对于输入 DNA 总量(等于 1)的信号进行表述。
Product Image 3: 5-Hydroxymethylcytosine (5-hmC) (HMC31) Mouse mAb Expand Image
5-Hydroxymethylcytosine (5-hmC) (HMC31) Mouse mAb 的特异性通过 DNA 免疫沉淀进行确定。对用小鼠胚胎干细胞制备且加入含有未甲基化胞嘧啶、5-甲基胞嘧啶 (5-mC) 或 5-羟甲基胞嘧啶 (5-hmC) 的 DNA 的基因组 DNA 进行 DNA 免疫沉淀。使用与内标的对照 DNA 序列特异的引物,通过实时 PCR 对富集的 DNA 进行定量分析。每种样品中免疫沉淀的 DNA 的量以相对于输入 DNA 总量(等于 1)的信号进行表述。