Assay of Ornithine Decarboxylase and Spermidine/Spermine N¹-acetyltransferase Activities

Materials and Reagents

1. EDTA (Sigma-Aldrich, catalog number: E9884 )
   
2. Tris-HCl (Sigma-Aldrich, catalog number: T5941 )
   
3. Triton X-100 (Sigma-Aldrich, catalog number: T8787 )
   
4. DL-dithiothreitol (DTT) (Sigma-Aldrich, catalog number: 43815 )
   
5. Protease inhibitor cocktail, such as Complete EDTA-free protease inhibitor tablets (Roche Diagnostics, catalog number: 11873580001 )
   
6. L-ornithine monohydrochloride (Sigma-Aldrich, catalog number: O2375 )
   
7. Acid-treated [¹⁴C]-L-ornithine (100 µCi/ml, 40-60 mCi/mmol) (PerkinElmer, catalog number: NEC710050UC )
   
8. Pyridoxal 5’phosphate monohydrate (PLP) (Sigma-Aldrich, catalog number: 82870 )
   
9. Citric acid (Sigma-Aldrich, catalog number: C0759 )
   
10. SOLVABLE (PerkinElmer, catalog number: 6NE9100 )
    
11. [Acetyl-1-¹⁴C]-Acetyl Coenzyme A (AcCoA) (20 µCi/ml, 40-60 mCi/mmol) (PerkinElmer, catalog number: NEC313050UC ) (Note 1)
    
12. Spermidine trihydrochloride (Sigma-Aldrich, catalog number: S2501 )
    
13. Hydroxylamine hydrochloride (Sigma-Aldrich, catalog number: 159417 )
    
14. ddH₂O
    
15. 96% ethanol such as ETAX A (Altia Oyj, catalog number: 12210143 )
    
16. Liquid scintillation cocktail, such as OptiPhase HiSafe 2 (PerkinElmer, catalog number: 1200-436 )
    
17. Buffer A (see Recipes)
    
18. 1 M Tris-HCl (see Recipes)
    
19. 200 mM EDTA (pH 8.0) (see Recipes)
    
20. 100 mM DTT (see Recipes)
    
21. Acid-treated [¹⁴C]-L-ornithine (see Recipes)
    
22. 10 mM or 25 mM L-ornithine (see Recipes)
    
23. 20 mM PLP (see Recipes)
    
24. 2 M citric acid (see Recipes)
    
25. 100 mM Spd (see Recipes)
    
26. 1 M hydroxylamine (see Recipes)
    

Equipment

1. 10-ml glass tubes with rubber caps (Note 2)
   
2. Tube rack for glass tubes
   
3. Whatman^TM 3MM Chr chromatography paper (Thermo Fisher Scientific, catalog number: 05-713-336 )
   Note: Cut into 3 cm x 2 cm size and folded 4 times (Figure 1A).
   
4. Long 18-21G needle
   
5. 10-ml syringe
   
6. Forceps
   
7. Tissue homogenizer, such as 3-ml Potter-Elvehjem glass tube and pestle (Sigma-Aldrich, catalog number: P7734 ) and a drill to operate the pestle
   
8. 0.5-ml microcentrifuge tubes
   
9. Microcentrifuge
   
10. Thermal cycler with block fitted for 0.5 ml tubes
    
11. Whatman^TM Grade P81 Ion Exchange Cellulose Chromatography Paper (Thermo Fisher Scientific, catalog number: 05-171-2A ) (cut into 1.5 x 1.5 cm size)
    
12. Parafilm^TM (Sigma-Aldrich, catalog number: P7793 )
    
13. Horizontal shaker
    
14. A sheet of filter paper
    
15. Wallac 4-ml plastic scintillation vials and caps (PerkinElmer, catalog number: 1200-421 )
    
16. Liquid scintillation counter and plates fitted for 4-ml vials
    

Procedure