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Histone methylation is an important epigenetic modification that plays important roles in plant development and growth. Histone methytransferases are the enzymes to establish histone methylation and here we describe a simple and effective protocol for detecting methyltransferase activity and specificity in vitro.

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Histone Methyltransferase Assay in vitro
组蛋白甲基转移酶活性体外检测试验

生物化学 > 蛋白质 > 活性
作者: Pengfei Sui
Pengfei SuiAffiliation: Department of Biochemistry, Fudan University, Shanghai, China
Bio-protocol author page: a197
Yu Yu
Yu YuAffiliation: Department of Biochemistry, Fudan University, Shanghai, China
Bio-protocol author page: a198
 and Aiwu Dong
Aiwu DongAffiliation: Department of Biochemistry, Fudan University, Shanghai, China
For correspondence: aiwudong@fudan.edu.cn
Bio-protocol author page: a199
Vol 2, Iss 24, 12/20/2012, 4518 views, 0 Q&A, How to cite
DOI: http://dx.doi.org/10.21769/BioProtoc.311

[Abstract] Histone methylation is an important epigenetic modification that plays important roles in plant development and growth. Histone methytransferases are the enzymes to establish histone methylation and here we describe a simple and effective protocol for detecting methyltransferase activity and specificity in vitro.

Keywords: Plant(计划), Histone H3 lysine 36(组蛋白H3赖氨酸36), Methyltransferase assay(甲基转移酶检测)

Materials and Reagents

  1. 14C labeled-methyl-adenosyl-L-methionine (SAMS) (Sigma-Aldrich)
  2. Purified recombinant histones (from E. coli)
  3. Histone H3 (Roche Diagnostics, catalog number: 1034758)
  4. Core histones (Sigma-Aldrich, catalog number: H9250)
  5. Mononuclosomes and Oligonucleosomes (see procedure)
  6. Coomassie brilliant blue R-250
  7. MiliQ water
  8. SDS-PAGE
  9. KCl
  10. MgCl2
  11. Sucrose
  12. ZnCl2
  13. β-mercaptoethanol
  14. MAB buffer (see Recipes)

Equipment

  1. Water bath

Procedure

  1. Substrate
    Purified recombinant Histones (Ding et al., 2007), histone H3, core histones, mononuclosome and oligonucleosome (from Hela cells or recombinant ones from E.coli).
    Note: The mononucleosomes and oligonucleosomes prepared from Hela cells were gifts from Yi Zhang (UNC Chapel Hill, USA). The recombinant oligonucleosomes from E. coli were gifts from Bing Zhu (NIBS, Beijing, China).

  2. Reaction
    A mixture containing 1-5 μl enzyme (such as SDG725), 5 μl 4x MAB buffer, 250 nCi 14C labeled SAM and 1 μg substrates to 20 μl volume, is kept at 37 °C for 1-2 h. The reaction is stopped by adding 5 μl 5x SDS-PAGE loading buffer and boiling at 100 °C for 5 min, and 5 μl samples will be analyzed by 15% SDS-PAGE gel electrophoresis and visualized by Coomassie brilliant blue R-250. The SDS-PAGE gel is dried and exposed to X-ray films or scanned by Typhoon (GE) (15% refers to the concentration of Acr/Bis. Different histones can be separated more clearly by 15% SDS-PAGE).

Recipes

  1. MAB buffer
    1x MAB buffer
    50 mM Tris-Cl (pH 8.5)
    20 mM KCl
    10 mM MgCl2
    250 mM Sucrose
    100 μM ZnCl2
    10 mM β-mercaptoethanol

Acknowledgments

The protocol was adapted from previously published papers (Rea et al., 2000; Ding et al., 2007).

References

  1. Ding, B., Zhu, Y., Gao J., Yu, Y., Cao, K. M., Shen W. H., Dong, A. (2007) Molecular characterization of three rice SET-domain proteins. Plant Sci 172:1072-1078.
  2. Rea, S., Eisenhaber, F., O'Carroll, D., Strahl, B. D., Sun, Z. W., Schmid, M., Opravil, S., Mechtler, K., Ponting, C. P., Allis, C. D. and Jenuwein, T. (2000). Regulation of chromatin structure by site-specific histone H3 methyltransferases. Nature 406(6796): 593-599.
  3. Sui, P., Jin, J., Ye, S., Mu, C., Gao, J., Feng, H., Shen, W. H., Yu, Y. and Dong, A. (2012). H3K36 methylation is critical for brassinosteroid-regulated plant growth and development in rice. Plant J 70(2): 340-347.


How to cite this protocol: Sui, P., Yu, Y. and Dong, A. (2012). Histone Methyltransferase Assay in vitro. Bio-protocol 2(24): e311. DOI: 10.21769/BioProtoc.311; Full Text



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