Comparative Physiology @ Guelph
Whole Troponin Exchange Protocol
500 mM 2,3-Butanedione Monoxime (BDM) (dissolve in ddw and pH to 7.0)
1 ml Prerigor solution + BDM (5 mM)
- 10 mM Imidozole
- 2.5 mM EGTA
- 15 mM EDTA
- pH 7 @ 15 oC
1 ml Rigor solution + BDM (5 mM)
- pCa 9 solution
- 5 mM EGTA
- 10 mM Imidizole
- pH 7.0 @ 15 oC
1 ml pCa 9.0 + BSA (1 mg/ ml)
- 100 mM KCl
- 10 mM MOPS
- 9 mM MgCl2
- 4 mM Na2-ATP
- 0.1 mM EGTA
- pH 7.0 @ 15 oC
pCa solutions (9.0-4.0)
150 ul Whole troponin solution (3-6 mg/ml)
Add before using: 1.5 ul BDM (500 mM stock)
1.5 ul DTT (100 mM stock)
1.5 ul protease inhibitor cocktail (from sigma)
- 1 Trabecule mounted, SL set to 2.3 and maximally activated to get pre-exchange force generation capability
- 2 Trabecule placed in rigor (30 min)
- 3 Whole troponin exchange (2 â€“3 hours)
- Set temperature of stage to 15 oC
- Mount trabeculae in pCa 9.0 solution then set SL to 2.3 and initiate Brenner cycle.
- Record FIS and HFS.
- Expose to pre-activation solution and then activate with pCa 4.5.
- Record ktr and HFS.
- Relax with pCa 9.
- Check SL
- Repeat activation
- Relax with pCa 9
- Stop the Brenner cycle
- Decease temperature to 5 oC
- Decrease SL to 2 or less (want to decrease tension on hooks)
- inject pre-rigor solution (+BDM)
- After 1 min inject rigor solution (+BDM)
- Turn off chart recorder
- Incubate for 30 min
- Using a pair of pliers cut tip off of 23 gauge needle and attach to 100 ul pippete (set to 45 ul)
- Increase temperature to 10 oC
- Remove cover slip and wash it in alcohol, then dry, and set aside
- Using kimwipe dry off top of chamber as well as area surrounding right side channel.
Pick up 45 ul of protein solution in pippete in preparation.
Using edge of kimwipe absorb all solution from inside the chamber by placing edge very close to the right side channel
Quickly pipette in 45 ul of protein solution on top of fiber then repeat. (need to make sure that there is enough solution in chamber that it will come in contact with coverslip and form a seal.
- Place on coverslip ensuring a seal. If it does not, carefully pipette in more without removing the cover slip.
- Turn off all lights and potential sources of heat.
- At 60 min, pipette in remaining protein solution.
- At 120 min or longer, wash in 1 ml of 9.0 solution + BSA.
- Increase temperature to 15 oC
Incubate 10 min.
Inject in pCa 9 solution and reset SL to 2.3. Activate to determine any loss of force. Please note that sometimes, depending on the proteins exchanged in a lower pCa is required to achieve maximal activation.