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Nuclear/Cytoplasmic Fractionation of Proteins from Caenorhabditis elegans

秀丽隐杆线虫中细胞核/细胞质蛋白的分离

AM Alejandro Mata-Cabana
Olga Sin Olga Sin
RS Renée I. Seinstra
EN Ellen A. A. Nollen

发布: 2018年10月20日第8卷第20期 DOI: 10.21769/BioProtoc.3053 浏览次数: 9890

评审: Matthias RieckherAnonymous reviewer(s)

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Abstract

C. elegans is widely used to investigate biological processes related to health and disease. To study protein localization, fluorescently-tagged proteins can be used in vivo or immunohistochemistry can be performed in whole worms. Here, we describe a technique to localize a protein of interest at a subcellular level in C. elegans lysates, which can give insight into the location, function and/or toxicity of proteins.

Keywords: C. elegans (秀丽隐杆线虫) search Subcellular fractionation (亚细胞分离) search Protein localization (蛋白定位) search Nucleus (细胞核) search Cytoplasm (细胞质) search Immunoblot (免疫印迹) search

Background

Subcellular fractionation has been used in different model organisms to identify and study protein function in nuclei, membranes and cytoplasm. For example, aggregation-prone proteins may be more toxic when they are localized in the nucleus or in the cytosol (Kontopoulos et al., 2006; Barmada et al., 2010). Here we provide a protocol (adapted from Chen et al., 2000 and La Rocca et al., 2007) to localize specific proteins in the nuclear and cytoplasmic fractions of C. elegans.

Materials and Reagents

  1. 94 mm plates (Greiner Bio One International, catalog number: 633185 )
  2. 15 ml conical tube (SARSTEDT, catalog number: 62.554.502 )
  3. 1.5 ml tubes (Greiner Bio One International, catalog number: 616201 )
  4. Pellet pestle (Sigma-Aldrich, catalog number: Z359947-100EA )
  5. Glass slides (Fisher Scientific, catalog number: 12164682 )
  6. Nitrocellulose (Bio-Rad Laboratories, catalog number: 1620112 )
  7. C. elegans strain
  8. Escherichia coli OP50 strain
  9. Deionized water (dH2O)
  10. Cholesterol (Fisher Scientific, catalog number: 10263660 )
  11. Ethanol absolute (Merck, catalog number: 1009831000 )
  12. Magnesium sulphate (MgSO4) (Fisher Scientific, catalog number: 10264630 )
  13. Calcium dichloride (CaCl2) (Fisher Scientific, catalog number: 10171800 )
  14. di-Potassium hydrogen phosphate (K2HPO4) (Merck, catalog number: 105101 )
  15. Potassium dihydrogen phosphate (KH2PO4) (Merck, catalog number: 104873 )
  16. Casein digest, Difco (BD, catalog number: 211610 )
  17. Select agar (Thermo Fisher Scientific, InvitrogenTM, catalog number: 30391049 )
  18. Disodium hydrogen phosphate (Na2HPO4) (Acros Organics, catalog number: 424380010 ) 
  19. Sodium chloride (NaCl) (Merck, catalog number: 106404 )
  20. DL-Dithiothreitol (DTT) (Sigma-Aldrich, catalog number: D0632 )
  21. HEPES (Sigma-Aldrich, catalog number: H4034 )
  22. Potassium hydroxide (KOH) (Fisher, catalog number: 10705921 )
  23. Potassium chloride (KCl) (Fisher Scientific, catalog number: 10010310 )
  24. Magnesium dichloride (MgCl2) (Fisher Scientific, catalog number: 10518060 )
  25. Ethylenediaminetetraacetic acid (EDTA) (Sigma-Aldrich, catalog number: E6758 )
  26. Sucrose (Fisher Scientific, catalog number: 10386100 )
  27. Tween 20 (Sigma-Aldrich, catalog number: P1379-100ML )
  28. Protease inhibitors (cOmplete) (Roche, catalog number: 11697498001 )
  29. Benzonase nuclease (Merck, catalog number: 70746 )
  30. PierceTM BCA Protein Assay Kit (Thermo Fisher Scientific, catalog number: 23225 )
  31. Liquid nitrogen
  32. Ammonium persulfate (APS) (Thermo Fisher Scientific, catalog number: A/6120/60 )
  33. N,N,N′,N′-Tetramethylethylenediamine (TEMED) (Fisher Scientific, catalog number: 10142863 )
  34. 40% Acrylamide/Bis solution (29:1) (Bio-Rad Laboratories, catalog number: 1610146 )
  35. Sodium dodecyl sulfate (SDS) (Merck, Calbiochem, catalog number: 428015 )
  36. Tris Base (Roche Diagnostics, catalog number: 11814273001 )
  37. Hydrochloric acid 37% (Acros Organics, catalog number: 124630010 )
  38. Glycine (Fisher Scientific, catalog number: 10070150 )
  39. Methanol (Merck, catalog number: 106009 )
  40. Bromophenol blue (Acros Organics, catalog number: 403160100 )
  41. Glycerol (Sigma-Aldrich, catalog number: G5516 )
  42. β-Mercaptoethanol (Merck, catalog number: 444203 )
  43. Milk powder (Campina)
  44. PageRulerTM Plus Prestained Protein Ladder (Thermo Fisher Scientific, catalog number: 26619 )
  45. Amersham ECL Prime Western Blotting Detection Reagent (GE Healthcare, catalog number: RPN2236 )
  46. Antibodies (see Table 1)

    Table 1. List of primary and secondary antibodies
    Primary antibody
    		Host
    		Company/Catalog number
    		Dilution primary antibody
    		Secondary antibody
    α-LMN-1
    		Rabbit
    		Novus Biologicals,
    catalog number:
    38530002
    		1:1,000
    		Anti-rabbit (1:10,000)
    (Bio-Rad Laboratories, catalog number: 1706515 )
    α-tubulin
    		Mouse
    		Sigma-Aldrich,
    catalog number:
    T6074
    		1:10,000
    		Anti-Mouse (1:10,000)
    (Bio-Rad Laboratories, catalog number: 1706516 )
    α-GFP 
    (Living Colors® A.v. Monoclonal Antibody (JL-8)
    		Mouse

    		Takara Bio,
    catalog number:
    632381
    		1:10,000
    		Anti-Mouse (1:10,000)
    (Bio-Rad Laboratories, catalog number: 1706516 )

  47. M9 buffer (see Recipes)
  48. Phosphate buffered saline (PBS) (see Recipes)
  49. PBS-T (see Recipes) 
  50. Blocking solution (see Recipes)
  51. Nematode growth medium (NGM) plates (see Recipes)
  52. Phosphate buffer (see Recipes)
  53. Hypotonic buffer (see Recipes)
  54. Hypertonic buffer (see Recipes)
  55. 25x protease inhibitors (see Recipes)
  56. 1 M DTT (see Recipes)
  57. 12% acrylamide gel (see Recipes)
  58. Running buffer (see Recipes)
  59. Transfer buffer (see Recipes)
  60. SDS lysis buffer (see Recipes)

Equipment

  1. Single channel pipettes (Gilson, models: P2G , P20G , P200G , P1000G )
  2. Orbital shaker (Thermo Fisher Scientific, model: MaxQTM 2000)
  3. 20 °C incubator (Winecooler, LIEBHERR, model: WK 4126 )
  4. Stereo microscope (Leica, model: MZ7.5 )
  5. Pellet pestle (motor) (Sigma-Aldrich, catalog number: Z359971 )
  6. Tabletop centrifuge, cooled (Eppendorf, model: 5424 R )
  7. Centrifuge (Thermo Fisher Scientific, model: SL 40R )
  8. Autoclave (VWR, model: VAPOUR-Line Lite )
  9. -80 °C freezer (Sanyo, model: VIP plus )
  10. Mini PROTEAN 3 system (Bio-Rad Laboratories, catalog number: 1658001edu )
  11. WB Imager (GE Healthcare, model: ImageQuant LAS 4000 mini, catalog number: 28955813 )

Software

  1. ImageJ (Open source: https://imagej.nih.gov/ij/)
  2. Microsoft Excel (Microsoft Corporation, Redmond, USA)

Procedure

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文章信息

版权信息

© 2018 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Mata-Cabana, A., Sin, O., Seinstra, R. I. and Nollen, E. A. A. (2018). Nuclear/Cytoplasmic Fractionation of Proteins from Caenorhabditis elegans. Bio-protocol 8(20): e3053. DOI: 10.21769/BioProtoc.3053.

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分类

生物化学 > 蛋白质 > 定量

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What is meant by washing the supernatant in step 4 of the procedure?
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