Western Analysis of Histone Modifications (Aspergillus nidulans)
Western检测组蛋白（巢状曲霉）的修饰
发布: 2013年04月05日第3卷第7期 DOI: 10.21769/BioProtoc.424 浏览次数: 12350
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参见作者原研究论文
The authors used this protocol in:
Oct 2012
Abstract
Western blotting allows for the specific detection of proteins and/or modifications of proteins by an antibody of interest. This protocol utilizes a crude nuclei extraction protocol for Aspergillus nidulans to enrich for histones and other nuclear proteins prior to gel electrophoresis. Post translational modifications of histones may then be easily detected. After electrophoresis, the selected antibodies are used to detect and quantify levels of the modifications of interest.
Keywords: Histone H4 (H4组)
Epigenetics (epigenetics)
Histone H3 (组蛋白H3)
Background
Materials and Reagents
- D-glucose
- NaNO3
- KCl
- MgSO4.7H2O
- KH2PO4
- ZnSO4.7H2O
- H3BO3
- MnCl2.4H2O
- FeSO4.7H2O
- CoCl2.5H2O
- CuSO4.5H2O
- (NH4)6Mo7O24.4H2O
- EDTA
- Spermine
- Spermidine
- PMSF
- Sorbitol
- Halt Protease Inhibitor Cocktail (Thermo Fisher Scientific, catalog number: PI-78437 )
- NaCl
- KCl
- Tris base
- Glycine
- Methanol
- Bio-Rad protein assay (Bio-Rad Laboratories, catalog number: 500-0006 )
- Miracloth (Calbiochem, catalog number: 475855 )
- Rabbit polyclonal antibody to histone H4 acetyl K5 (1:2,500) (Abcam, catalog number: ab51997 )
- Rabbit polyclonal antibody to histone H4 acetyl K8 (1:2,500) (Abcam, catalog number: ab15823 )
- Rabbit polyclonal antibody to human C-terminus histone H4 antibody (1:2,500) (Abcam, catalog number: ab10158 )
- Mouse monoclonal antibody to TATA Binding Protein (TBP) (1:1,000) (Abcam, catalog number: ab61411 )
- Mouse monoclonal antibody to pan acetyl H4 (1:1,000) (Active Motif, catalog number: 39925 )
- Goat anti-mouse alkaline phosphatase conjugated antibody (1:5,000) (Life Technologies, Gibco®, catalog number: 13864-012 )
- Goat anti-rabbit alkaline phosphatase conjugated antibody (1:5,000) (Pierce Antibodies, catalog number: 31342 )
- 1-Step NBT/BCIP (Thermo Fisher Scientific, catalog number: 34042 )
- Glucose Minimal Medium (GMM) (see Recipes)
- 2x Laemmli buffer (see Recipes)
- Nuclear Isolation buffer (see Recipes)
- Resuspension buffer (see Recipes)
- ST buffer (see Recipes)
- TBS-T (see Recipes)
- Western transfer buffer (see Recipes)
- Media Recipes (see Recipes)
Equipment
- Mortar and pestle
- Bio-Rad Mini-Protean gel electrophoresis system (Bio-Rad Laboratories, catalog number: 170-3940 )
- Semi-Dry Blotter (Bio-Rad Laboratories, catalog number: 170-3940)
- Orbital shaker
- 15 ml Falcon tubes
- 50 ml Falcon tubes
- 30 ml centrifuge tubes
- Microfuge tubes
Procedure
文章信息
版权信息
© 2013 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Soukup, A. and Keller, N. P. (2013). Western Analysis of Histone Modifications (Aspergillus nidulans). Bio-protocol 3(7): e424. DOI: 10.21769/BioProtoc.424.
Download Citation in RIS Format
分类
分子生物学 > 蛋白质 > 检测
生物化学 > 蛋白质 > 免疫检测 > 免疫印迹法(WB )
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