ATPase

   HOME     RESEARCH     PUBLICATIONS     PROTOCOLS     CONTACT    

ATPase solutions

see Kirk Lab Protocol One Drive

Force-ATPase Protocol_Tom Lynch

Day 1

• 1. Make 1X KHB from 10X KH Buffer stock in H20
• 2. Make relaxing solution (can freezer O/N for Day 2)
• 3. Make permeabilization solution
• 4. Take out mouse heart and perfuse through aorta with KHB
• 5. Bring heart to SLAB stereoscope
• 6. Pin down the heart at the right and left atria and apex, and cut through aorta down septum to expose RV
• 7. Remove pin at apex and cut down the septum
• 8. Peel open the LV
• 9. You will see 2 large trunks, these are the papillary muscles
• 10. Slide forceps under papillary muscles
• 11. Cut papillary muscles as high and as low as you can perpendicular to the fiber direction
• 12. Cut the top of the fiber and then cut down to the bottom of the fiber and cut the bottom of the fiber
• 13. Put the fiber in the permeabilization solution O/N
• 14. Can keep the rest of the heart for further experiments if needed in -80

Day 2

• 1. Obtain two 35mm dish cap and a metal plate on ice
• 2. Thaw reagents (in -20C #1)
  • a. Relax solution made on Day 1
  • b. Green labeled tubes = activating solution
  • c. Blue labeled tubes = preactivating solution
  • d. Red labeled tubes = relaxing solution
  • e. ADP clear tube = ADP
  • f. Black tube = NADH
• 3. After reagents are thawed, add 100 ul of LDH 1 mg/ml (0.1 mg/ml total), 100 ul ddH2O and 1 ug PK (use Pak lab scales, it is more accurate) into all the solutions and vortex well. You can use frozen LDH for couple of weeks.
• 4. Spin down solutions at 7000 g for 2 mins at 4 degrees.
• 5. Obtain a #10 scalpel blade
• 6. Obtain surgical tools and T clips
• 7. NEXT…
• 8. Place 35mm dish cap under stereoscope
• 9. Put scalpel blade in holder with plyers
• 10. On scope, set stereoscopic vision by zooming all the way in, focusing, and zooming back out
• 11. Obtain another 35mm dish cap and and label date, genotype and animal ID
• 12. Pour large relax solution into 35mm dish cap
• 13. Take the permeabilization tube from the fridge post-O/N incubation
• 14. Remove solution and replace with fresh relax solution with a small transfer pipette
• 15. Put fiber in dish with transfer pipette and bring into focus (fiber will appear somewhat balled up)
• 16. Check the quality of the fibers
• 17. You can check that by placing light on the same plane as the dish cap so that light passes through the fibers and not on the top
• 18. When finding a good fiber, split along the fiber parallel, make sure you anchor the blade in the dish cap before splitting and cut like a paper cutter
• 19. Trim excess off of fiber along the damaged edge
• 20. Do a visual inspection of the fiber
• 21. Remove some of the excess and the terminal ends of the fiber and discard the pieces
• 22. Transfer the fibers to the labeled dish cap
• 23. Put dish cap with fibers on the metal plate on ice
• 24. Next, remove a T-clip from sheet by gently pulling it off
• 25. Transfer cut T-clips to dish with fibers and push the T-clips into the solution
• 26. Under stereoscope, flip up the wings of the T-clips
• 27. Place fiber on both clips
• 28. Close wings around fiber on both clips relatively simultaneously
• 29. Gently press wings in the middle and four edges to secure the fiber
• 30. Wash tools with H20 and move to the Force-ATPase system

Force-ATPase Assay

• 1. Turn on all 7 on switches (computer, main ATPase power, force transducer, spectrophotometer light, mixer, water bath, stereoscope light)
• 2. Clean large relax chamber with H20 using transfer pipette
• 3. Clean relax chamber and preactivating chamber with 170 ul of H20
• 4. Place stiring syringe with bubble on the mixer
• 5. Fill ADP syringe (red) with ADP and load onto the injector
• 6. Clean activating chamber with 20 ul H20 using H20 syringe ( large clear syringe)
• 7. Place circular stand in hole on table and put on dish with T-clipped fiber
• 8. Attach the fiber to the length controller first and then the force transducer
• 9. Move the fiber to the large relax chamber and set the sarcomere length to 2 um using the laser diffraction
• 10. Clean all syringes
• 11. When filling syringes, first pipette up a small amount and suck through the syringe and then dispense to clean out syringe
• 12. Add about 170 ul relax and preactivating solutions into appropriate chambers with 200 ul pipette
• 13. Using the ruler, measure fiber length in the large relaxing solution and record length on excel sheet
• 14. Move the fiber into the relax solution and wait 3 minutes
• 15. After the 3 minutes, move the fiber to the preactivating solution for at least 3 minutes
• 16. During this time, rinse the activating chamber 3 times with activating solution using the activating syringe (green) making sure to oscillate during washes and then turn off the oscillation when removing solution for the next wash
• 17. Leave the third wash in the chamber and go to the NADH program
• 18. Hit the arrow on the program and then hit “do it AH”
• 19. Add a very small drop of NADH from NADH syringe (small clear syringe) into activating solution and get the 340nm value to match the 20% 340nm value (± 0.01)
• 20. After setting the number, hit the stop circle button
• 21. Next, hit the “auto zero” red button on the force machine above the Force-ATPase set-up
• 22. Next, go to the force measurement program and hit the “run” arrow, then “Zero NADH”, then “reset”
• 23. When the NADH line stabilizes, hit “Zero NADH” and “reset” again
• 24. Then, move the fiber into the activating solution
• 25. When the force stabilizes, type the force value into the excel sheet
• 26. Then, move the fiber into the large relax solution for 1 minute
• 27. After 1 minute, step on the ADP pedal 3 times quickly
• 28. Then hit “Zero NADH”
• 29. When it stabilizes hit the pedal again, and then 3 more times for a total of 4 times
• 30. When finished, hit the large “stop” circle button, write data to file
• 31. Set the sarcomere length again with the laser (can move laser if needed)
• 32. Measure muscle length, width and depth (with mirror) in 3 locations and record values if they are changed
• 33. SYSTEM HAS NOW BEEN CALIBRATED, NOW START EXPERIMENT
• 34. Move fiber into small relax solution chamber for at least 3 minutes
• 35. Then, move fiber to preactivating solution for at least 3 minutes
• 36. During this time, rinse out the activating solution 5X with dH20 and then 3X with 100% activating solution
• 37. Leave the third wash in the chamber and go to the NADH program
• 38. Hit the arrow on the program and then hit “do it AH”
• 39. Add a very small drop of NADH from NADH syringe (small clear syringe) into activating solution and get the 340nm value to match the 20% 340nm value (± 0.01)
• 40. After setting the number, hit the stop circle button
• 41. Next, go to the force measurement program and hit the “run” arrow, then “Zero NADH”, then “reset”
• 42. When the NADH line stabilizes, hit “Zero NADH” and “reset” again
• 43. Then, move the fiber into the activating solution
• 44. When the force stabilizes, type the force value into the excel sheet
• 45. Then, move the fiber into the small relax solution for 1 minute
• 46. After 1 minute, step on the ADP pedal 2 times quickly
• 47. Then hit “Zero NADH”
• 48. When it stabilizes hit the pedal again, and then 4 more times for a total of 5 times (steps) at 10-15 second intervals
• 49. When finished, hit the large “stop” circle button, write data to file
• 50. Repeat from step 35 through all Ca2+ solutions
• 51. The final activation will be back at 100% to check run-down, if more than 20% run-down, fiber is no good
• 52. To measure Ktr, after checking run down, clean system as before with H20 and 100% activating solution
• 53. Activate force to max as before
• 54. When force is at its max, turn black knob to right to Ktr
• 55. Turn OFF the oscillator
• 56. Hit “stop” on the program
• 57. Open the “shortcut to Ktr program”
• 58. On the window on the left
• 59. Change window to 3000
• 60. Input muscle length from white box
• 61. Hit the check mark
• 62. Then hit the run arrow, Ktr graph should appear
• 63. Then save
• 64. TURN Ktr BACK TO SL!!!!
• 65. EXPERIMENT IS COMPLETE FOR THIS FIBER
• 66. Dismount
• 67. Move force-transduer/length controller all the way to the left and clean all baths and syringes 5X with dH20
• 68. Turn off all 8 switches
• 69. Save data on USB