Density Gradient Centrifugation for Enrichment and Identification of GFP-tagged Chitosomal Microvesicles of Filamentous Fungi

Materials and Reagents

1. Tygon R-3603 tubing, I.D. 1/16 inch (Saint-Gobain, catalog number: AAC00002 )
2. Grade 1 Qualitative Filter Papers (GE Healthcare, catalog number: 1001-090 )
   
3. Neurospora crassa conidia
   
4. Sucrose (Sigma-Aldrich, catalog number: S0389 )
   
5. Sodium phosphate monobasic (NaH₂PO₄^.H₂O) (Thermo Fisher Scientific, Fisher Scientific, catalog number: S369-500 )
   
6. Sodium phosphate dibasic anhydrous (Na₂HPO₄) (Sigma-Aldrich, catalog number: S-7907 )
   
7. Protease Inhibitor Cocktail, Complete ULTRA tablets EDTA-free (Roche Diagnostics, catalog number: 04693132001 )
   Note: Currently, it is “Sigma-Aldrich, catalog number: 04693132001 ”.
   
8. Glass beads (500 μm) (Bio Spec Product, catalog number: 11079105 )
   
9. 10% (w/v) sucrose in 33 mM (final) phosphate buffer (pH 8.2) (steam sterilize and store at 4 °C until use)
   
10. 65% (w/v) sucrose in 33 mM (final) phosphate buffer (pH 8.2) (steam sterilize and store at 4 °C until use)
    
11. D-Biotin (Faga Labs, catalog number: CAS-58-85-5 )
    
12. Vogel’s complete medium (see Recipes)
    
13. 50x salt solution (see Recipes)
    
14. Trace element solution (see Recipes)
    
15. 100 mM phosphate buffer (pH 8.2) (see Recipes)
    
16. Laemmli buffer (see Recipes)
    

Equipment

1. Braun MSK 50 ml shaking bottles (LABEQUIP LTD, catalog number: 8541302 )
2. VWR^® Standard Hot Plate Stirrers (VWR International, catalog number: 12365-382 )
3. Quick-seal centrifuge tube (Beckman Coulter, catalog number: 344326 )
   
4. Shaker incubator (Lab-LineX, model: Orbit Environ Shaker )
   
5. Vacuum filtration system
   
6. Braun MSK cell homogenizer (LABEQUIP LTD, catalog number: 953030 )
   
7. Bright field microscope (Olympus, model: Vanox-S )
   
8. Beckman L8-70M Ultracentrifuge (pre-cooled at 4 °C) (Beckman Coulter)
   
9. Type 70Ti rotor (pre-cooled at 4 °C) (Beckman Coulter)
   
10. GM-40 Linear Gradient maker (C. B. S. Scientific)
    
11. Peristaltic (Multi-staltic) pump (Buchler Lab, catalog number: N/A )
    
12. Tube sealer (Beckman Coulter, catalog number: 342420 )
    
13. Density gradient fractionator (ISCO, 185)
    Note: This model is not available anymore. Brandel BR-186 Gradient Fractionator with Syringe Pump can be used instead.
    
14. CIGNET^TM Fraction collector (ISCO)
    Note: This item is also discontinued, but Bio-Rad 2110 Fraction Collector can be used instead.
    
15. 6505 W UV-Vis Spectrophotometer (Bibby-scientific, Jenway)
    
16. Refractometer (ZEISS, catalog number: 12230 )
    

Procedure