ATPase assay for dynein (radioactive)

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[MATERIALS] - [INDEX]

Procedure

1. Mix:
   
   • 30 µl sample
   • 10 µl PEM or MT (for MT activated activity)
   • 10 µl 5X ATP mix
   • 50 µl total
   • 
     
   Set up a MT blank if doing MT activated activity assay:
   
   • 30 µl PEM
   • 10 µl MT
   • 10 µl 5X ATP mix
   • 50 µl total
   • 
     
2. Incubate at room temperature for 20 min.
3. 
   
4. Stop reaction with 300 µl acid.
5. 
   
6. Add 300 µl charcoal (stirred).
7. 
   
8. Incubate on ice 10 min.
9. 
   
10. Add 100 µl BSA.
11. 
    
12. Centrifuge (cold) 1 minute.
13. 
    
14. Count 0.3 ml supernate.
    
    To calculate activity:
    [sample cpm - blank cpm- MT blank if appropriate] x [ratio: total assay volume/assay volume counted] x [1 nmol/cpm/nmol] x [1/ sample volume] x [1/assay time]
    
    For example, for a 20 min assay, with 30 µl sample, counting 300 µl out of 750 µl total volume:
    [sample cpm - blank cpm] x 1.5 x 1 nmol/cpm/nmol x 1/0.03 ml x 1/20 min= nmol Pi/ml/min
15. 
    

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Materials

• 20 mM silicotungstic acid (Baker, MW 2882) in 10 mM H₂SO₄
• 
  
• 25% suspension of acid washed charcoal (Sigma)
• 
  
• 5 mg/ml BSA in H₂O
• 
  
• 5X ATP mix
  
  • 5 mM ATP
  • 5 mM MgSO₂
  • 1 µl ³²P-ATP
• 
  
• 5X MT (@ 3 mg/ml tubulin + taxol, warm briefly)

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