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In vitro Histone H3 Cleavage Assay for Yeast and Chicken Liver H3 Protease

体外组蛋白H3裂解分析法测定酵母和鸡肝H3蛋白酶的活性

SC Sakshi Chauhan
GA Gajendra Kumar Azad
RT Raghuvir Singh Tomar

发布: 2017年01月05日第7卷第1期 DOI: 10.21769/BioProtoc.2085 浏览次数: 9926

评审: Yanjie LiDamián Lobato-MárquezAnonymous reviewer(s)

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Abstract

Histone proteins are subjected to a wide array of reversible and irreversible post-translational modifications (PTMs) (Bannister and Kouzarides, 2011; Azad and Tomar, 2014). The PTMs on histones are known to regulate chromatin structure and function. Histones are irreversibly modified by proteolytic clipping of their tail domains. The proteolytic clipping of histone tails is continuously attracting interest of researchers in the field of chromatin biology. We can recapitulate H3-clipping by performing in vitro H3 cleavage assay. Here, we are presenting the detailed protocol to perform in vitro H3 cleavage assay.

Keywords: Histone H3 (组蛋白H3) search Chicken liver H3 protease (鸡肝H3蛋白酶) search Yeast H3 protease (酵母H3蛋白酶) search Histone clipping (组蛋白剪切) search Chromatin (染色质) search

Background

Histone H3 clipping is the least understood mechanism of chromatin modification and regulation. It is expected that H3 clipping will permanently erase PTMs from the nucleosomes that might affect chromatin related events. Moreover, the fate of cleaved histones is still under investigation and it has been suggested that the cleaved histones might be recycled at specific regions of chromatin or they are targeted for degradation. There are various reports that describe in vivo clipping of histone H3 in different organisms, while in vitro assays for histone H3-specific clipping are limited. We need an efficient and robust in vitro assay for characterizing histone specific proteases. To this end, we present a protocol that can be used to examine the in vitro histone H3 clipping activity of yeast and chicken liver histone H3 proteases. We have optimized temperature and pH conditions for the assay. Under our optimized conditions, proteases were found to specifically cleave histone H3 out of all core histones. We have extensively used this protocol in our recent publications (Chauhan et al., 2016; Chauhan and Tomar, 2016; Azad and Tomar, 2016; Mandal et al., 2014; Mandal et al., 2013; Mandal et al., 2012). This protocol can be used to identify and characterize histone H3 specific proteases from different organisms ranging from yeast to mammals.

Materials and Reagents

  1. 1.5 ml centrifuge tubes (Tarsons)
  2. Dialysis tubing cellulose membrane (Sigma-Aldrich, catalog number: D9277 )
  3. Amicon ultra centrifugal filters (EMD Millipore, catalog number: UFC900396 )
  4. Cheese cloth
  5. Freshly extracted or frozen chicken brain tissue
  6. Saccharomyces cerevisiae yeast cells (BY4743 strain)
  7. Freshly extracted liver tissue from chicken
  8. Triton X-100 (Sigma-Aldrich, catalog number: T8787 )
  9. Sucrose (Sigma-Aldrich, catalog number: S7903 )
  10. Sodium chloride (NaCl) (EMD Millipore, catalog number: 567440 )
  11. Acetone (HiMedia Laboratories, catalog number: AS024 )
  12. Lyticase (Sigma-Aldrich, catalog number: L4025 )
  13. Protein A sepharose bead (GE Healthcare, catalog number: 17-0963-03 )
  14. Ammonium persulfate (Sigma-Aldrich, catalog number: A3678 )
  15. Ammonium sulfate [(NH4)2SO4] (Sigma-Aldrich, catalog number: A2939 )
  16. Coomassie Brilliant Blue R (Sigma-Aldrich, catalog number: B0149 )
  17. Potassium chloride (KCl) (EMD Millipore, catalog number: 104936 )
  18. Spermidine (Sigma-Aldrich, catalog number: S2626 )
  19. Spermine (Sigma-Aldrich, catalog number: 85590 )
  20. EDTA (Sigma-Aldrich, catalog number: EDS )
  21. EGTA (Sigma-Aldrich, catalog number: E3889 )
  22. β-mercaptoethanol (Sigma-Aldrich, catalog number: M3148 )
  23. Phenylmethanesulfonyl fluoride (PMSF) (Sigma-Aldrich, catalog number: 7626 )
  24. Disodium hydrogen phosphate (EMD Millipore, catalog number: 106586 )
  25. Sodium dihydrogen phosphate (EMD Millipore, catalog number: 106370 )
  26. HEPES (Sigma-Aldrich, catalog number: H3375 )
  27. Glycerol (EMD Millipore, catalog number: 104093 )
  28. Protease inhibitor cocktail (PIC) (Sigma-Aldrich, catalog number: P2714 )
  29. Potassium acetate (Sigma-Aldrich, catalog number: P1190 )
  30. DTT (Sigma-Aldrich, catalog number: D9779 )
  31. Sorbitol (Sigma-Aldrich, catalog number: S6021 )
  32. Magnesium chloride hexahydrate (MgCl2) (Sigma-Aldrich, catalog number: 63064 )
  33. Sodium dodecyl sulfate (SDS) (Sigma-Aldrich, catalog number: L3771 )
  34. Trizma base (Sigma-Aldrich, catalog number: T6066 )
  35. Hydroxyapatite resin (Sigma-Aldrich, catalog number: 289396 )
  36. Sulfuric acid (H2SO4) (EMD Millipore, catalog number: 112080 )
  37. Acrylamide (Sigma-Aldrich, catalog number: A8887 )
  38. GLUD1 antibody (Sigma-Aldrich, catalog number: SAB2100932-50UG )
    Note: This Product has been discontinued.
  39. Solution 1 (see Recipes)
  40. Hypotonic solution (see Recipes)
  41. Sodium phosphate buffer (1 M), pH 6.8 (see Recipes)
  42. HB buffer (see Recipes)
  43. HAP buffer (see Recipes)
  44. Potassium acetate solution (see Recipes)
  45. Prespheroplasting buffer (see Recipes)
  46. Spheroplasting buffer (see Recipes)
  47. Wash buffer (see Recipes)
  48. Lysis buffer (see Recipes)
  49. Protein A sepharose beads (50% slurry) (see Recipes)
  50. Yeast-protease resuspension buffer (see Recipes)
  51. Protease dialysis buffer (see Recipes)
  52. Reaction buffer
    1. Yeast protease reaction buffer (see Recipes)
    2. Chicken liver protease reaction buffer (see Recipes)
  53. 10% Triton X-100 solution (see Recipes)
  54. 6x SDS-PAGE loading dye (see Recipes)
  55. 10x SDS-PAGE running buffer (see Recipes)

Equipment

  1. Homogenizer (Pro Scientific, model: Bio-Gen PRO200 homogenizer )
  2. Potter-Elvehjem PTFE pestle and glass tube (Sigma-Aldrich, catalog number: P7734 )
  3. Centrifuge (Beckman Coulter, models: Avanti® J-E centrifuge-floor , Microfuge® 22R centrifuge )
  4. Sonicator (Boston Industries, model: Branson Digital Sonifier 250 & 102C Converter Ultrasonic Dismembrator )
    Note: This product has been sold out.
  5. Ultracentrifuge (Beckman Coulter, model: OptimaTM L-100K ultracentrifuge )
  6. Bio-Rad electrophoresis power supply (Bio-Rad Laboratories, model: PowerPacTM Basic Power Supply )
  7. Bio-Rad SDS-PAGE running apparatus (Bio-Rad Laboratories, model: Mini-PROTEAN tetra cell )
  8. Shaker (Eppendorf, model: New BrunswickTM Innova® 44 )
  9. Rotamer (Tarsons, catalog number: 3090 )
  10. Heating block, Thermocell Cooling and heating Block (Bio-Equip, model: HB-202 )
  11. FPLC cold cabinet (UniEquip, model: Unichromat 1500 )
  12. SuperoseTM 6, 10/300 GL chromatography column (GE Healthcare, catalog number: 17-5172-01 )

Procedure

English
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文章信息

版权信息

© 2017 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Chauhan, S., Azad, G. K. and Tomar, R. S. (2017). In vitro Histone H3 Cleavage Assay for Yeast and Chicken Liver H3 Protease. Bio-protocol 7(1): e2085. DOI: 10.21769/BioProtoc.2085.

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分类

癌症生物学 > 癌症生物化学 > 蛋白质

生物化学 > 蛋白质 > 活性

生物化学 > 蛋白质 > 分离和纯化

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