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Cycloheximide Assays to Measure Protein Degradation in vivo in Plants

体内放线菌酮实验测定植物中的蛋白质降解

Jonathan Gilkerson Jonathan Gilkerson
RT Raymond Tam
AZ Aimee Zhang
KD Kate Dreher
JC Judy Callis

发布: 2016年09月05日第6卷第17期 DOI: 10.21769/BioProtoc.1919 浏览次数: 20695

评审: Tie LiuYuko KuritaAnonymous reviewer(s)

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参见作者原研究论文

The authors used this protocol in:

Cover of Plant Physiology, featuring study using the protocol.

Jun 2015

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Abstract

The half-life of a protein is a characteristic property, and can be modulated by post-translational modifications, changes in subcellular localization, and/or interaction with other proteins or ligands. As one determinant of its steady-state level, a protein’s degradation represents an important distinguishing attribute relevant to its biological function. Because protein longevity cannot be elucidated from bioinformatics analyses, it must be determined empirically. Here we describe two approaches for in vivo half-life determination in plants: 1. pooled-seedling degradation assays monitoring either tagged versions of the protein (luciferase fusions or other epitope tags) or following the endogenous protein; 2. single-seedling degradation assays using luciferase fusion proteins. The advantages of these approaches are their simplicity and low cost.

Keywords: Protein degradation (蛋白质的降解) search Cycloheximide (放线菌酮) search Proteolysis (蛋白水解) search Half-life (半衰期) search Degradation assays (降解实验) search

Materials and Reagents

  1. 60 x 15 mm sterile Petri dishes (Corning, Falcon®, catalog number: 353002 )
  2. Microfuge tubes
  3. Pipet tips
  4. White polystyrene flat-bottom 96-well plates (Thermo Fisher Scientific, Fisher Scientific, catalog number: 12-566-619 )
  5. PVDF (polyvinylidene fluoride) Western protein blotting membrane (Sigma-Aldrich, Amersham HybondTM, catalog number: GE10600023 )
  6. Clear adhesive plate film (USA-Scientific, catalog number: 2921-7810 )
  7. X-ray film (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 34091 )
  8. Plant lines expressing protein of interest, either from transgene or from endogenous locus
  9. Cycloheximide (CHX) (Sigma-Aldrich, catalog number: C1988 )
  10. Primary antibody [example, anti-HA-HRP (3F10) (Sigma-Aldrich, Roche, catalog number: 12158167001 )]
  11. Secondary antibody (horseradish peroxidase-linked) (Bio-Rad Laboratories, catalog number: 1662408EDU )
  12. Tween-20 (Sigma-Aldrich, catalog number: P9416 )
  13. Bleach (sodium hypochlorite) (VWR International, Chlorox®, catalog number: 89501-620 ) or from a grocery or drugstore, such as Chlorox bleach
  14. Murashige and Skoog basal salts with micronutrients (Sigma-Aldrich, catalog number: M5524 )
  15. Sucrose
  16. MES [2-(N-morpholino)ethanesulfonic acid] free acid (Merck Millipore, Calbiochem®, catalog number: 475893 )
  17. Nicotinic acid (Sigma-Aldrich, catalog number: N4126 )
  18. Thiamine-HCl (Sigma-Aldrich, catalog number: T4625 )
  19. Pyroxidine-HCl (Sigma-Aldrich, catalog number: P9755 )
  20. Myo-inositol (Sigma-Aldrich, catalog number: I1525 )
  21. Potassium hydroxide (KOH) (Thermo Fisher Scientific, Fisher Scientific, catalog number: P250 )
  22. Bacto agar (BD, Difco, catalog number: 214010 )
  23. Parafilm (Thermo Fisher Scientific, Fisher Scientific, catalog number: 13-374-10 )
  24. Liquid nitrogen
  25. Potassium phosphate monobasic (KH2PO4) (Sigma-Aldrich, catalog number: P9791 )
  26. Potassium phosphate dibasic (K2HPO4) (Sigma-Aldrich, catalog number: P3786 )
  27. Ethylenediaminetetraacetic acid (EDTA), disodium dehydrate (Thermo Fisher Scientific, catalog number: S311 )
  28. β-mercaptoethanol (Sigma-Aldrich, catalog number: M6250 )
  29. Phenylmethylsulfonyl fluoride (PMSF) (Sigma-Aldrich, catalog number: P7626 )
  30. Tricine (Sigma-Aldrich, catalog number: T0377 )
  31. Magnesium chloride hexahydrate (MgCl2) (Thermo Fisher Scientific, Fisher Scientific, catalog number: M33 )
  32. BSA (bovine serum albumin) for protein standard curve (Sigma-Aldrich, catalog number: A7030 )
  33. Adenosine 5’-triphosphate disodium salt hydrate (ATP) (Sigma-Aldrich, catalog number: A2383 )
  34. Tris base (Sigma-Aldrich, catalog number: T1503 )
  35. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S3014 )
  36. IGEPAL CA-630 [Octylphenoxy poly (ethyleneoxy) ethanol] (Sigma-Aldrich, catalog number: I8896 ), a nonionic, non-denaturing detergent replacement for Nonidet P-40
  37. MG132 (Benzyloxycarbonyl-L-Leucyl-L-Leucyl-L-Leucinal) (Peptides International, catalog number: IZL-3175-v )
  38. Complete mini protease inhibitor EDTA-free tablet (Sigma-Aldrich, Roche, catalog number: 4693159001 )
  39. Protein concentration assay (Bio-Rad Laboratories, catalog number: 5000006 )
  40. Chemiluminescent western blot development kit (SuperSignal West Pico or Dura Extended duration substrate) (Thermo Fisher Scientific, catalog number: 34077 and 34075 , respectively)
  41. D-Luciferin potassium salt (Gold Biotechnology, catalog number: LUCK-100 )
  42. Growth media (GM) (see Recipes)
  43. Luciferase extraction buffer (see Recipes)
  44. Luciferase assay buffer (see Recipes)
  45. Protein extraction buffer for Western blot assays (see Recipes)

Equipment

  1. Plate reading spectrophotometer for protein determination assay (Thermo Fisher Scientific)
  2. Refrigerated centrifuge (Eppendorf)
  3. SDS-PAGE gel apparatus and power supply (Thermo Fisher Scientific)
  4. Porcelain mortar and pestle for protein extraction (Thermo Fisher Scientific)
  5. Western transfer apparatus (Thermo Fisher Scientific)
  6. Plate reading luminometer (Berthold Technologies)
  7. Flatbed scanner (Hewlett-Packard)
  8. X-ray film developer, either a set of tanks or trays, or an automatic machine
  9. Orbital shaker for Western blot incubation (Thermo Fisher Scientific)

Software

  1. Excel and another statistical package such as GraphPad Prism or Stata
  2. NIH ImageJ 1.36 (http://rsb.info.nih.gov/ij/)
  3. ImageQuant 1.0 software (http://www.gelifesciences.com/webapp/wcs/stores/servlet/catalog/en/GELifeSciences-us/products/AlternativeProductStructure_16016/29000605)

Procedure

English
中文翻译

文章信息

版权信息

© 2016 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Readers should cite both the Bio-protocol article and the original research article where this protocol was used:

  1. Gilkerson, J., Tam, R., Zhang, A., Dreher, K. and Callis, J. (2016). Cycloheximide Assays to Measure Protein Degradation in vivo in Plants. Bio-protocol 6(17): e1919. DOI: 10.21769/BioProtoc.1919.
  2. Gilkerson, J., Kelley, D. R., Tam, R., Estelle, M. and Callis, J. (2015). Lysine residues are not required for proteasome-mediated proteolysis of the auxin/indole acidic acid protein IAA1. Plant Physiology 168(2): 708-720.

    3. ris ris Download Citation in RIS Format

分类

植物科学 > 植物生物化学 > 蛋白质

生物化学 > 蛋白质 > 降解

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