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Chase Assay of Protein Stability in Haloferax volcanii

沃氏嗜盐富饶菌中蛋白质稳定性的追踪测定

Xian Fu Xian Fu
Julie A. Maupin-Furlow Julie A. Maupin-Furlow

发布: 2017年03月20日第7卷第6期 DOI: 10.21769/BioProtoc.2186 浏览次数: 6731

评审: Yanjie LiLyu XiaomeiHonghong Wu

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Abstract

Highly regulated and targeted protein degradation plays a fundamental role in almost all cellular processes. Determination of the protein half-life by the chase assay serves as a powerful and popular strategy to compare the protein stability and study proteolysis pathways in cells. Here, we describe a chase assay in Haloferax volcanii, a halophilic archaeon as the model organism.

Keywords: Archaea (古生菌) search Ubiquitin-proteasome system (泛素 - 蛋白酶体系统) search Targeted proteolysis (靶向蛋白水解) search SAMP (SAMP) search Chase assay (追踪测定) search

Background

In eukaryotes, the ubiquitin proteasome system plays a major role in highly selective and targeted proteolysis (Glickman and Ciechanover, 2002). Recent evidence shows that small archaeal ubiquitin-like modifier proteins or SAMPs also function in targeting proteins for destruction by proteasomes (Maupin-Furlow, 2014; Anjum et al., 2015; Fu et al., 2016). Measurement of the protein half-life in vivo provides a direct way to study the proteolysis pathway. Cycloheximide chase and pulse-chase assays are commonly used to monitor the degradation of the targeted protein in eukaryotes (Zhou, 2004). The former method is utilized to determine the half-life of all cellular proteins after inhibition of translation elongation by cycloheximide; whereas, the pulse-chase assay measures the turnover of newly synthesized (pulse-labeled) proteins without interfering the normal cell growth. Compared with the eukaryotic system, a rapid and simple method to determine the half-life of a given protein in archaea is not well established. Therefore, we developed a protocol to measure the protein stability in vivo for the salt-loving archaeon Haloferax volcanii. Inhibitors of translation (anisomycin) and transcription (actinomycin D) are utilized to minimize the synthesis of new protein in this archaeon. TBP2, a TATA-binding protein (TBP) modified by ubiquitin-like isopeptide bonds in Haloferax volcanii, serves as the model protein substrate in this study.

Materials and Reagents

  1. Sterilized wooden stick
  2. Parafilm
  3. Zip-lock plastic bags
  4. 13 x 100 mm2 culture tubes (Fisher Scientific, catalog number: 14-961-27 )
  5. 1.5 ml microcentrifuge tube (Fisher Scientific, catalog number: 02-681-320 )
  6. Polyvinylidene difluoride (PVDF) membrane (GE Healthcare, catalog number: 10600023 )
  7. X-ray film (RPI, catalog number: 248300 )
  8. 2.0 ml microcentrifuge tube (Fisher Scientific, catalog number: 02-681-321 )
  9. Disposable plastic cuvettes (Fisher Scientific, catalog number: 149-551-27 )
  10. Gloves (Fisher Scientific, catalog number: 19-130-1597C )
  11. Rack LTS tips
    ‘P20’ 2-20 µl (Mettler-Toledo, Rainin, catalog number: 17001865 )
    ‘P200’ 20-200 µl (Mettler-Toledo, Rainin, catalog number: 17001863 )
    ‘P1000’ 100-1,000 µl (Mettler-Toledo, Rainin, catalog number: 17001864 )
  12. Sterile polystyrene disposable serological 10 ml pipets with magnifier stripe (Fisher Scientific, catalog number: 13-678-11E )
  13. Nalgene rapid-flow sterile disposable bottle top 0.2 µm filters with surfactant-free cellulose acetate (SFCA) membrane (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 290-3320 )
  14. Haloferax volcanii cells (parent and ubiquitin-like proteasome system mutants) carrying the reporter plasmid pJAM2201 encoding Flag-SAMP2 and TBP2-StrepII under control of the P2rrnA constitutive promoter. Plasmid pJAM202c served as the empty vector control. Please refer to strain and plasmid information published as Table S2 in (Fu et al., 2016)
  15. Glycerol (Sigma-Aldrich, catalog number: G5516 )
  16. Agar (Sigma-Aldrich, catalog number: A7002 )
  17. Novobiocin (Sigma-Aldrich, catalog number: N1628 )
  18. Actinomycin D (Sigma-Aldrich, catalog number: A1410 )
  19. Anisomycin (Sigma-Aldrich, catalog number: A9789 )
  20. Acetone (Sigma-Aldrich, catalog number: 650501 )
  21. Methanol (Fisher Scientific, catalog number: A413 )
  22. CDP-Star (Thermo Fisher Scientific, Molecular ProbesTM, catalog number: T2304 )
  23. Coomassie brilliant blue R-250 staining solution (Bio-Rad Laboratories, catalog number: 1610436 )
  24. Antibodies
    1. Anti-StrepII polyclonal antibody (in mouse) (QIAGEN, catalog number: 34850 )
    2. Goat anti-mouse IgG (whole molecule)-alkaline phosphatase-linked antibody (Sigma-Aldrich, catalog number: A5153 )
    3. Alkaline phosphatase-linked anti-Flag M2 monoclonal antibody (Sigma-Aldrich, catalog number: A9469 )
  25. Sodium chloride (NaCl) (Fisher Scientific, catalog number: S642-12 )
  26. Magnesium chloride hexahydrate (MgCl2·6H2O) (Fisher Scientific, catalog number: M35-12 )
  27. Potassium sulfate (K2SO4) (Fisher Scientific, catalog number: P304-3 )
  28. Calcium chloride dihydrate (CaCl2·2H2O) (Fisher Scientific, catalog number: C79-500 )
  29. Tryptone (BD, BactoTM, catalog number: 211705 )
  30. Yeast extract (BD, BBL, catalog number: 211929 )
  31. Deionized H2O
  32. Sodium hydroxide (NaOH) (Fisher Scientific, catalog number: BP359-212 )
  33. Magnesium sulfate heptahydrate (MgSO4·7H2O) (Fisher Scientific, catalog number: M63-3 )
  34. Potassium chloride (KCl) (Fisher Scientific, catalog number: P217-3 )
  35. Tris-base (Fisher Scientific, catalog number: BP152-1 )
  36. Sodium dodecyl sulfate (SDS) (Fisher Scientific, catalog number: BP166-500 )
  37. Glycine (Bio-Rad Laboratories, catalog number: 1610718 )
  38. β-mercaptoethanol (Sigma-Aldrich, catalog number: M6250 )
  39. Bromophenol blue (Sigma-Aldrich, catalog number: B5525 )
  40. Acrylamide (30%) (Fisher Scientific, catalog number: EC890450ML )
  41. ATCC974 medium (see Recipes)
  42. Concentrated salt water (SW) stock solution at 30% (w/v) (see Recipes)
  43. 2x SDS reducing buffer (see Recipes)

Equipment

  1. Incubator and shaker (42 °C) (Eppendorf, New Brunswick Scientific)
  2. SmartSpec Plus spectrophotometer (Bio-Rad Laboratories, catalog number: 1702525 )
    Note: This product has been discontinued.
  3. Centrifuge (Eppendorf, model: 5418 )
  4. Basic power supply (Bio-Rad Laboratories, catalog number: 1645050 )
  5. Refrigerator (4 °C) (Frigidare)
  6. Ultra-low temperature freezer (-80 °C) (Eppendorf, New BrunswickTM, model: C660-86 )
  7. Vortex mixer (Thermolyne, model: 37600 )
  8. Chemical fume hood (Siemens, catalog number: 537-473 )
  9. Pipettes (2-20 µl, 20-200 µl, 100-1,000 µl) (Rainin type LTS)
  10. pH meter (Corning, model: 320 )
  11. Scanner (Epson, model: 3170 Photo )
  12. Autoclave (Consolidated Sterilizer Systems, model: SR-24C-PB )
  13. Mini trans-blot module (Bio-Rad Laboratories, catalog number: 1703935EDU )
  14. Gel electrophoresis chamber (Bio-Rad Laboratories, catalog number: 1658005 )
  15. Konica X-ray film processor (Konica Minolta, model: QX60A )
  16. Electrophoresis systems autoradiography cassette (Fisher Scientific, FisherBiotech, model: FBXC810 )
  17. Siemens Vantage Reverse Osmosis Systems (M21 series, Siemens, model: M21R004EA ) with EVOQUA filters (Siemens, model: C1207098 ) and Atlantic Ultraviolet Germicidal UV Equipment (Siemens, model: MP49 ) (water purification system used for generating deionized water)

Software

  1. ImageJ (imagej.net/Particle_Analysis)
  2. Microsoft Excel (Microsoft Office 365 ProPlus)

Procedure

English
中文翻译

文章信息

版权信息

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

如何引用

Fu, X. and Maupin-Furlow, J. A. (2017). Chase Assay of Protein Stability in Haloferax volcanii. Bio-protocol 7(6): e2186. DOI: 10.21769/BioProtoc.2186.

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微生物学 > 微生物生物化学 > 蛋白质

生物化学 > 蛋白质 > 降解

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