Stem Microsome Preparation and Mannan Synthase Activity Assay

Materials and Reagents

1. cOmplete, Mini, EDTA-free Protease Inhibitor Cocktail Tablets (Roche, catalog number: 11836170001 )
   
2. GDP-[¹⁴C]-Man (9.694 GBq/mmol, 262.0 mCi/mmol, 76.34 μM) (PerkinElmer, catalog number: NEC536050UC )
   
3. Bicinchoninic acid (BCA) Protein Assay Kit (Pierce Antibodies, catalog number: 23225 or 23227 )
   
4. OptiPhase Supermix Cocktail (PerkinElmer, catalog number: 1200-439 )
   
5. GDP-Man (Sigma-Aldrich, catalog number: G-7377 )
   
6. Carob galactomannan (Megazyme International, catalogue number: P-GALML )
   
7. Sucrose
   
8. MgCl₂
   
9. Extraction buffer (EB) (see Recipes)
   
10. 4x ManS assay buffer (see Recipes)
    
11. 1 mM GDP-Man (see Recipes)
    
12. 1% carob galactomannan (see Recipes)
    

Equipment

1. Mortar and pestle
   
2. 13 ml Sarstedt tube (Sarstedt, catalog number: 60.540.500 )
   
3. Centrifuge
   
4. 4 ml plastic liquid scintillation counting vial (PerkinElmer, catalog number: 1200-421 )
   
5. 1450 MicroBeta TriLux Microplate Scintillation and Luminescence Counter (PerkinElmer, catalog number: 1450-024 )
   

Procedure