CTPase assay for dynein (colorimetric)

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[MATERIALS] - [INDEX]

Procedure

1. Make standard curve: 0, 5, 10, 15, 20, 25, 30 µl phosphate solution in 100 µl H₂O.
2. 
   
3. Prepare samples:
   
   
   • Make - MT/CTP blank (subtract from basal activity samples):
     
     • 40 µl H₂O
     • 40 µl P100
     • 10 µl PEM
     • 10 µl CTP mix (add last, right before incubation)
     • 
       
   • Make + MT/CTP blank (subtract from microtubule-activated samples)
     
     • 40 µl H₂O
     • 40 µl P100
     • 10 µl MT mix
     • 10 µl CTP mix (add last, right before incubation)
     • 
       
   • Make sample blanks (subtract from appropriate samples):
     
     • 50 µl H₂O
     • 40 µl each sample
     • 10 µl PEM
     • 
       
   • Basal activity samples:
     
     • 40 µl H₂O
     • 40 µl each sample
     • 10 µl PEM
     • 10 µl CTP mix (add last)
     • 
       
   • MT activated activity samples:
     
     • 40 µl H₂O
     • 40 µl each sample
     • 10 µl MT mix
     • 10 µl CTP mix (add last)
     • 
       
   • 
     
4. Incubate all tubes at 37°C, 30 minutes exactly. Start incubation immediately after adding CTP mix to tubes.
5. 
   
6. Stop each reaction with 900 µl STOP mix.
   Note: Make STOP mix fresh and use immediately. Stop mix should be very light yellow. If stop mix is greenish, do not use--make sure reagents are in Milli-Q water and make fresh.
7. 
   
8. To develop, incubate stopped reactions 20 min at 45°C or 60 min at 37°C or 90 min at room temp.
9. 
   
10. Read absorbances at 820 nm.
11. 
    
12. Calculate specific activities. Subtract sample blanks and CTP blanks from appropriate reactions first.
    
    SA= (corrected abs) x (1/slope of std. curve) x (1/min) x (1/ml) x (1/mg/ml protein)= nmol Pi/min/mg
    
    For example: for a 30 min assay time, 40 µl samples with 0.04 mg/ml protein, and a std curve slope of 0.02:
    SA= (corrected abs) x (1/.02) x (1/30min) x (1/.04 ml) x (1/.04)
13. 
    

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Materials

• 1 mM phosphate solution for standard curve (1 L)
  
  • 0.136 g KH₂PO₄ in H₂O
  
  
• CTP mix
  
  • 10 mM CTP (sodium salt) in 10 mM MgCl₂
• 
  
• Ammonium Molybdate (1 L)
  
  • 4.2 g molybdic acid
  • 28.6 ml concentrated H₂SO₄
• 
  
• Ascorbate
  
  • 13% ascorbic acid in H₂O
• 
  
• 10% SDS (use extra-pure stuff)
• 
  
• MT mix
  
  • 5-10 mg/ml DEAE tubulin with 10 mM taxol, warm briefly, make fresh
• 
  
• STOP mix (45 ml)
  
  • 30 ml molybdic acid stock
  • 5 ml ascorbic acid stock
  • 2.5 ml 10 % SDS
  • 7.5 ml Milli-Q water
  • 
    

Make all solutions with Milli-Q water!

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