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Immunoblot Analysis of Histone H4 Acetylation and Histone H2A Phosphorylation in Candida albicans
白色念珠菌中组蛋白H4乙酰化和组蛋白H2A磷酸化的免疫印迹分析   

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Abstract

Posttranslational modifications of histones are required for different processes including transcription, replication and DNA damage repair. This protocol describes the preparation of a whole-cell extracts for the fungal pathogen Candida albicans. Furthermore, the extract is used to detect lysine acetylation of histone H4 as well as serine 129 phosphorylation of histone H2A by immunoblot analysis.

Keywords: Histone(组蛋白), Acetylation(乙酰化), Phosphorylation(磷酸化), Candida albicans(白色念珠菌)

Materials and Reagents

  1. Candida albicans
  2. TCA (trichloroacetic acid) (Merck KGaA, catalog number: 641730 )
  3. Recombinant histone H4 (New England Biolabs, catalog number: M2504S )
  4. Calf histones (Sigma-Aldrich, catalog number: H9250 )
  5. 0.033% sodium azide (Merck KGaA, catalog number: 8223350250 )
  6. Nitrocellulose membrane (Millipore, catalog number: Protran BA79)
  7. Whatman filter paper 3 MM Chr (Whatman, catalog number: 3030-917 )
  8. BSA (PAA Laboratories GmbH, catalog number: K41-001 )
  9. Sodium azide (Merck KGaA, catalog number: 822335 )
  10. Rabbit polyclonal antibody to histone H4 acetyl K5 (Abcam, catalog number: ab51997 ) (dilution: 1:5,000)
  11. Rabbit polyclonal antibody to histone H4 acetyl K8 (Active Motif, catalog number: 39172 ) (dilution: 1:3,000)
  12. Rabbit polyclonal antibody to histone H4 acetyl K12 (Millipore, catalog number: 07-959 ) (dilution: 1:3,000)
  13. Rabbit polyclonal antibody to histone H4 C-terminus (Abcam, catalog number: ab10158 ) (dilution: 1:1,000)
  14. Rabbit polyclonal antibody to histone H2A phospho-serine 129 (Active Motif, catalog number: 39271 ) (dilution: 1:2,000)
  15. Rabbit polyclonal antibody to histone H2A (Active Motif, catalog number: 39236 ) (dilution: 1:5,000)
  16. IRDye 800CW goat anti-rabbit IgG (H + L) (LI-COR, catalog number: 926-32211 )
  17. IRDye 680RD goat anti-rabbit IgG (H + L) (LI-COR, catalog number: 926-68071 )
  18. 4.5% stacking gel
  19. 20% running gel
  20. Bacto Yeast Extract (Becton, Dickinson and Company, catalog number: 212720 )
  21. Bacto Peptone (Becton, Dickinson and Company, catalog number: 211820 )
  22. Glucose (Merck KGaA, catalog number: 346351 )
  23. β-Mercaptoethanol (Sigma-Aldrich, catalog number: M3148-100ML )
  24. Urea (Sigma-Aldrich, catalog number: U5378-1KG )
  25. SDS (AppliChem GmbH, catalog number: A1112,1000 )
  26. EDTA (Sigma-Aldrich, catalog number: E5134-1KG )
  27. Bromphenolblue
  28. YPD medium (see Recipes)
  29. Yex lysis buffer (see Recipes)
  30. Protein sample buffer (see Recipes)
  31. SDS-PAGE running buffer (see Recipes)
  32. Running buffer (see Recipes)
  33. Transfer buffer (see Recipes)
  34. TBS and TBS-T (see Recipes)
  35. PBS (see Recipes)

Equipment

  1. Mini-Protean gel electrophoresis system (Bio-Rad, model: 165-8000 )
  2. Mini Trans-Blot cell (Bio-Rad, model: 170-3930 )
  3. 1.5 ml microcentrifuge tubes
  4. 15 ml Falcon tubes
  5. 14 ml Snap-cap tubes
  6. Eppendorf Thermomixer comfort (Eppendorf, model: 5355 000.011 )
  7. Shaking incubator
  8. Orbital shaker
  9. LI-COR Odyssey CLx Infrared scanner (LI-COR, model: P/N 9140-WP )
  10. CASY Cell Counter Model TT (Roche, catalog number: 05651735001 )

Software

  1. LI-COR Odyssey CLx imaging system

Procedure

  1. Whole-cell extract preparation
    1. Inoculate a single colony in 5 ml of YPD medium in a 14 ml Snap-cap tube and grow overnight at 30 °C shaking with 220 rpm.
    2. Dilute overnight culture to an OD600 of 0.1 in 5 ml YPD medium in a 14 ml Snap-cap tube and grow cells to OD600 of 1 at 30 °C shaking with 220 rpm.
      Note: For strains/conditions with different cell morphologies determine cell number by CASY measurement according to the CASY operator manual and use a total number of 5 x 107 cells. Flasks can also be used instead of Snap-cap tubes.
    3. Harvest cells by centrifugation in 15 ml Falcon tubes for 3 min at 1,500 x g.
    4. Resuspend pellet in 1 ml ice-cold H2O.
      Note: Work on ice from now on.
    5. Transfer suspension to 1.5 ml tube.
    6. Add 150 μl ice-cold Yex lysis buffer, vortex thoroughly and incubate on ice for 10 min.
    7. Add 150 μl ice-cold 50% (w/v) TCA and incubate on ice for 10 min to precipitate proteins.
    8. Spin for 5 min at 10,000 x g 4 °C and discard supernatant with a pipette.
    9. Spin for 1 min at 10,000 x g 4 °C and remove rest of the supernatant.
    10. Resuspend pellet in 100 μl protein sample buffer.
      Note: Resuspend by pipetting; if pH turns acidic (protein sample buffer turns yellow), add 1 M Tris base to increase pH (until sample buffer turns blue again).
    11. Incubate at 37 °C for 15 min shaking at 900 rpm.
    12. Spin down cell debris at 10,000 x g for 5 min and use 10 μl (0.5 OD600 units) of the supernatant for SDS-PAGE.

  2. SDS-PAGE and western blotting
    1. Load samples on a polyacrylamid gel (4.5% stacking gel and 20% running gel).
    2. Load 0.5 μg recombinant histone H4 in protein sample buffer as negative control and 2 μg calf histones in protein sample buffer as positive control for acetylation analysis.
      Note: Gel cast as described previously (Sambrook and Russell, 2001); Bio-Rad Mini-Protean gel electrophoresis system was used.
    3. Run gel at 150 V.
    4. Transfer proteins to nitrocellulose membrane by electroblotting using the Bio-Rad Mini Trans-Blot cell.
      Assemble blotting cassette in transfer buffer according to the Mini Trans-Blot instruction manual and run at 200 mA for 1 h at room temperature.
    5. Block membrane for 1 h at room temperature on an orbital shaker using 5% (w/v) BSA in TBS.
    6. Incubate with primary antibody diluted in TBS-T overnight at 4 °C on an orbital shaker.
    7. Pour off primary antibody solution and wash 3 x 10 min in TBS-T at room temperature.
      Note: Primary antibody solution can be reused several times (depending on the antibody); if solution is reused, add 0.033% sodium azide as preservative.
    8. Incubate with secondary antibody diluted 1:10,000 in TBS-T for 45 min at room temperature on an orbital shaker.
      Note: IRDye 800CW or IRDye 680RD secondary antibodies can be used.
    9. Pour off secondary antibody solution and wash 3 x 10 min in TBS-T at room temperature.
    10. Rinse blot briefly with PBS and scan using the LI-COR Odyssey CLx Infrared scanner.
      Note: Scanner settings: Intensity: 5; Resolution: 168 μm; Quality: medium.

Recipes

  1. YPD medium
    10 g/L Bacto Yeast Extract
    20 g/L Bacto Peptone
    20 g/L Glucose (add after autoclaving as 10x stock)
  2. Yex lysis buffer
    1.85 M NaOH
    7.5% (v/v) β-Mercaptoethanol (freshly added)
  3. Protein sample buffer
    40 mM Tris-HCl, pH 6.8
    8 M Urea
    5% (w/v) SDS
    0.1 mM EDTA
    1% (v/v) β-Mercaptoethanol (freshly added)
    0.1 g/L Bromphenolblue
  4. Running buffer
    25 mM Tris
    192 mM Glycine
    0.1% (w/v) SDS
  5. Transfer buffer
    25 mM Tris
    192 mM Glycine
    20% (v/v) Methanol
  6. TBS
    25 mM Tris
    140 mM NaCl
    2.5 mM KCl
    pH 7.4
  7. TBS-T
    TBS with 0.1% (v/v) Tween 20
  8. PBS
    140 mM NaCl
    2.5 mM KCl
    8.1 mM Na2HPO4
    1.5 mM KH2PO4
    pH 7.3

Acknowledgments

We thank all laboratory members for helpful discussions. This work was supported by a grant from the Christian Doppler Society, and in part by a grant from the Austrian Science Foundation (Project FWF-P-25333), to K.K. M.T. was supported through the Vienna Biocenter PhD Programme WK001.

References

  1. Sambrook, J. & Russell, D.W. (2001). Molecular Cloning, Volume 3, 3rd edition, Cold Spring Harbor Laboratory Press.
  2. Tscherner, M., Stappler, E., Hnisz, D. and Kuchler, K. (2012). The histone acetyltransferase Hat1 facilitates DNA damage repair and morphogenesis in Candida albicans. Mol Microbiol 86(5): 1197-1214.

简介

包括转录,复制和DNA损伤修复的不同过程需要组蛋白的翻译后修饰。 该方案描述了真菌病原体白色念珠菌的全细胞提取物的制备。 此外,提取物用于通过免疫印迹分析检测组蛋白H4的赖氨酸乙酰化以及组蛋白H2A的丝氨酸129磷酸化。

关键字:组蛋白, 乙酰化, 磷酸化, 白色念珠菌

材料和试剂

  1. 白色念珠菌
  2. TCA(三氯乙酸)(Merck KGaA,目录号:641730)
  3. 重组组蛋白H4(New England Biolabs,目录号:M2504S)
  4. 小牛组蛋白(Sigma-Aldrich,目录号:H9250)
  5. 0.033%叠氮化钠(Merck KGaA,目录号:8223350250)
  6. 硝化纤维素膜(Millipore,目录号:Protran BA79)
  7. Whatman滤纸3MM Chr(Whatman,目录号:3030-917)
  8. BSA(PAA Laboratories GmbH,目录号:K41-001)
  9. 叠氮化钠(Merck KGaA,目录号:822335)
  10. 针对组蛋白H4乙酰基K5的兔多克隆抗体(Abcam,目录号:ab51997)(稀释度:1:5,000)
  11. 针对组蛋白H4乙酰基K8的兔多克隆抗体(Active Motif,目录号:39172)(稀释度:1:3,000)
  12. 针对组蛋白H4乙酰基K12的兔多克隆抗体(Millipore,目录号:07-959)(稀释度:1:3,000)
  13. 兔多克隆抗体组蛋白H4 C末端(Abcam,目录号:ab10158)(稀释度:1:1,000)
  14. 针对组蛋白H2A磷酸丝氨酸129的兔多克隆抗体(Active Motif,目录号:39271)(稀释度:1:2,000)
  15. 针对组蛋白H2A的兔多克隆抗体(Active Motif,目录号:39236)(稀释度:1:5,000)
  16. IRDye 800CW山羊抗兔IgG(H + L)(LI-COR,目录号:926-32211)
  17. IRDye 680RD山羊抗兔IgG(H + L)(LI-COR,目录号:926-68071)
  18. 4.5%堆积凝胶
  19. 20%运行凝胶
  20. Bacto酵母提取物(Becton,Dickinson and Company,目录号:212720)
  21. Bacto蛋白胨(Becton,Dickinson and Company,目录号:211820)
  22. 葡萄糖(Merck KGaA,目录号:346351)
  23. β-巯基乙醇(Sigma-Aldrich,目录号:M3148-100ML)
  24. 尿素(Sigma-Aldrich,目录号:U5378-1KG)
  25. SDS(AppliChem GmbH,目录号:A1112,1000)
  26. EDTA(Sigma-Aldrich,目录号:E5134-1KG)
  27. 溴酚蓝
  28. YPD介质(参见配方)
  29. Yex裂解缓冲液(参见配方)
  30. 蛋白质样品缓冲液(参见配方)
  31. SDS-PAGE运行缓冲液(参见配方)
  32. 运行缓冲区(参见配方)
  33. 传输缓冲区(请参阅配方)
  34. TBS和TBS-T(参见配方)
  35. PBS(请参阅配方)

设备

  1. Mini-Protean凝胶电泳系统(Bio-Rad,型号:165-8000)
  2. Mini Trans-Blot细胞(Bio-Rad,型号:170-3930)
  3. 1.5 ml微量离心管
  4. 15 ml Falcon管
  5. 14 ml Snap-cap管
  6. Eppendorf Thermomixer comfort(Eppendorf,型号:5355 000.011)
  7. 摇动培养箱
  8. 轨道振动器
  9. LI-COR Odyssey CLx红外扫描仪(LI-COR,型号:P/N 9140-WP)
  10. CASY细胞计数器模型TT(Roche,目录号:05651735001)

软件

  1. LI-COR Odyssey CLx成像系统

程序

  1. 全细胞提取物制备
    1. 在14ml Snap-cap管中接种在5ml YPD培养基中的单个菌落,并在30℃下以220rpm振荡生长过夜。
    2. 在14ml Snap-cap管中,在5ml YPD培养基中将过夜培养物稀释至OD 600为0.1,并在30℃振荡下使细胞生长至OD 600 600。 220 rpm。
      注意:对于具有不同细胞形态的菌株/条件,根据CASY操作者手册通过CASY测量确定细胞数,并使用总数为5×10 7个细胞。 也可以使用烧瓶代替快速管。
    3. 通过在15ml Falcon管中在1,500×g离心3分钟收获细胞
    4. 将沉淀重悬在1ml冰冷的H 2 O中 注意:从现在开始在冰上工作。
    5. 将悬浮液转移至1.5ml管
    6. 加入150μl冰冷的Yex裂解缓冲液,彻底涡旋并在冰上孵育10分钟
    7. 加入150μl冰冷的50%(w/v)TCA,在冰上孵育10分钟以沉淀蛋白质。
    8. 在10,000×g/4℃下旋转5分钟,并用移液管丢弃上清液。
    9. 在10,000×g/4℃下旋转1分钟,并除去剩余的上清液
    10. 在100μl蛋白质样品缓冲液中重悬沉淀 注意:通过移液器重悬; 如果pH变为酸性(蛋白质样品缓冲液变黄),加入1M Tris碱以增加pH(直到样品缓冲液再次变蓝)。
    11. 在37℃下孵育15分钟,在900rpm下摇动
    12. 在10,000×g下旋转细胞碎片5分钟,并使用10μl(0.5OD 600单位)的上清液用于SDS-PAGE。

  2. SDS-PAGE和western印迹
    1. 将样品装在聚丙烯酰胺凝胶(4.5%堆积凝胶和20%流动凝胶)上
    2. 在蛋白质样品缓冲液中加载0.5μg重组组蛋白H4作为阴性对照,在蛋白质样品缓冲液中加入2μg小牛组蛋白作为乙酰化分析的阳性对照。
      注意:如前所述的凝胶模型(Sambrook和Russell,2001);使用了Bio-Rad Mini-Protean凝胶电泳系统。
    3. 在150 V下运行凝胶。
    4. 通过使用Bio-Rad Mini Trans-Blot细胞的电印迹将蛋白转移到硝酸纤维素膜上 根据Mini Trans-Blot说明手册在转移缓冲液中装配吸印盒,并在室温下以200 mA运行1小时。
    5. 在使用5%(w/v)BSA的TBS中的轨道振荡器上室温封闭膜1小时。
    6. 与在TBS-T中稀释的一抗在4℃下在轨道摇床上孵育过夜
    7. 倾倒一抗溶液,在室温下在TBS-T中洗涤3×10分钟 注意:一次抗体溶液可以重复使用几次(取决于抗体); 如果溶液重复使用,加0.033%叠氮化钠作为防腐剂。
    8. 用在TBS-T中1:10,000稀释的二抗在室温下在定轨振荡器上孵育45分钟。
      注意:可以使用IRDye 800CW或IRDye 680RD二抗。
    9. 倒出第二抗体溶液,在室温下在TBS-T中洗涤3×10分钟
    10. 用PBS短暂冲洗污渍,并使用LI-COR Odyssey CLx红外扫描仪扫描 注意:扫描仪设置:强度:5; 分辨率:168μm; 质量:中等。

食谱

  1. YPD介质
    10 g/L细菌酵母提取物
    20g/L Bacto蛋白胨 20 g/L葡萄糖(高压灭菌后加入10x原液)
  2. Yex裂解缓冲液
    1.85 M NaOH
    7.5%(v/v)β-巯基乙醇(新鲜加入)
  3. 蛋白质样品缓冲液
    40mM Tris-HCl,pH 6.8
    8 M尿素
    5%(w/v)SDS
    0.1mM EDTA
    1%(v/v)β-巯基乙醇(新鲜加入)
    0.1g/L溴酚蓝
  4. 运行缓冲
    25 mM Tris
    192 mM甘氨酸 0.1%(w/v)SDS
  5. 传输缓冲区
    25 mM Tris
    192 mM甘氨酸 20%(v/v)甲醇
  6. TBS
    25 mM Tris
    140mM NaCl 2.5mM KCl
    pH 7.4
  7. TBS-T
    TBS和0.1%(v/v)吐温20
  8. PBS
    140mM NaCl 2.5mM KCl
    8.1mM Na 2 HPO 4
    1.5mM KH 2 PO 4 4/v/v pH 7.3

致谢

我们感谢所有实验室成员进行有益的讨论。 这项工作得到了基督教多普勒学会的资助,部分得到了奥地利科学基金会(项目FWF-P-25333),K.K.的资助。 公吨。 通过维也纳生物中心博士课程WK001支持。

参考文献

  1. Sambrook,J。& 罗素 (2001)。 Molecular Cloning,第3卷,第3版,Cold Spring Harbor Laboratory Press
  2. Tscherner,M.,Stappler,E.,Hnisz,D。和Kuchler,K。(2012)。 组蛋白乙酰转移酶Hat1有助于白色念珠菌中的DNA损伤修复和形态发生。 Mol Microbiol 86(5):1197-1214。
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免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Tscherner, M. and Kuchler, K. (2013). Immunoblot Analysis of Histone H4 Acetylation and Histone H2A Phosphorylation in Candida albicans. Bio-protocol 3(20): e943. DOI: 10.21769/BioProtoc.943.
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