Detection of Hog1 Phosphorylation in Candida albicans in Response to an Antifungal Protein
白色念珠菌中响应抗真菌蛋白Hog1磷酸化水平的检测
发布: 2014年09月20日第4卷第18期 DOI: 10.21769/BioProtoc.1244 浏览次数: 9525
评审: Anonymous reviewer(s)
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参见作者原研究论文
The authors used this protocol in:
Aug 2013
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Abstract
It is becoming increasingly apparent that stress signalling is important for tolerance of fungal species to antifungal chemicals and proteins. The high-osmolarity glycerol (HOG) pathway responds to a number of stressors including osmotic and oxidative stress. This protocol describes a method to detect activation of the Candida albicans (C. albicans) MAPK Hog1 by monitoring its phosphorylation in response to an antifungal protein.
Materials and Reagents
- Candida albicans wild-type (WT)
- C. albicans hog1Δ/Δ deletion mutant
- Half-strength potato dextrose broth (½ PDB) (BD, DifcoTM, catalog number: 254920 )
- Protein of interest (e.g. the plant defensin NaD1)
- 5 M NaCl
- Ultrapure ice-cold water
- Dry ice
- 20% trichloroacetic acid (TCA) (20% w/v)
- 10% trichloroacetic acid (TCA) (10% w/v)
- 100% ice-cold acetone
- 4x NuPAGE LDS sample buffer (Life Technologies, catalog number: NP0007 )
- Bond-Breaker TCEP solution (neutral pH) (Thermo Fisher Scientific, catalog number: 77720 )
- 1 M Tris (pH 8.0)
- Any kD Mini-PROTEAN TGX precast polyacrylamide gels (15 well) (Bio-Rad Laboratories, catalog number: 456-9036 )
- 10x Tris/glycine/SDS running buffer (Bio-Rad Laboratories, catalog number: 161-0072 )
- 5% (w/v) skim milk in TBS
- Phospho-p38 MAPK (Thr180/Tyr182) antibody (Cell Signaling Technology, catalog number: 9211 )
- Hog1 (y-215) antibody (Santa Cruz, catalog number: sc-9079 )
- Amersham ECL rabbit IgG (HRP-linked antibody from donkey) (GE, catalog number: NA934 )
- Amersham ECL Western blotting detection reagents (GE, catalog number: RPN2106 )
- Yeast peptone dextrose broth (YPD) (see Recipes)
- Yeast peptone dextrose agar (YPD agar) (see Recipes)
- Tris-buffered saline (TBS) (see Recipes)
- Tris-buffered saline with Tween 20 (TBST) (see Recipes)
Equipment
- 180 μm glass beads (acid-washed) (Sigma-Aldrich, catalog number: G1152 )
- Trans-blot turbo mini PVDF transfer pack (Bio-Rad Laboratories, catalog number: 170-4156 )
- 50 ml tubes screw-cap tubes
- 30 °C shaking incubator
- Spectrophotometer
- 500 ml baffled flask
- Eppendorf 5810 R centrifuge (Eppendorf, catalog number: 5811 000.010 )
- 1.5 ml microfuge tubes
- Vortex
- Bench top centrifuge
- Trans-blot turbo transfer system (Bio-Rad Laboratories, catalog number: 170-4155 )
- Circular mixer
- Bio-Rad ChemiDoc MP imaging system (Bio-Rad Laboratories, catalog number: 170-8280 )
Procedure
文章信息
版权信息
© 2014 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Hayes, B. M. and Weerden, N. L. V. D. (2014). Detection of Hog1 Phosphorylation in Candida albicans in Response to an Antifungal Protein. Bio-protocol 4(18): e1244. DOI: 10.21769/BioProtoc.1244.
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分类
微生物学 > 抗微生物试验 > 抗真菌试验
微生物学 > 微生物信号传导 > 磷酸化
生物化学 > 蛋白质 > 免疫检测 > 免疫印迹法(WB )
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