Comparative Physiology @ Guelph
Chemistry For Stopped Flow Machine
Note: Always use the same (left) syringe housing for protein.
Use ABS at 280 with appropriate extinction coefficient to calculate protein concentration
To calculate extinction coefficient, use the Peptide Properties Calculator.
| Protein | Extinction Coefficeint |
| WT Rabbit sTnC | 2680 |
| WT Rat cTnC | 4080 |
| F27W rabbit sTnC | 8370 |
| F27W rabbit cTnC | 9770 |
For example, if OD280 for F27W cTnC is 1.8062, % by extinction coefficient (9770)
To calculate M concentration: 1.8062 % 9770= 0.000185 M = 185uM
To Use Tryptophan Fluorescence to Measure Ca2+ Offrate, set up for reaction is:
| Left Syringe | Right Syringe |
| 10-90 buffer plus 1mM DTT | 10-90 buffer plus 1mM DTT |
| 200 uM Ca2+ | 10 mM EGTA |
| 6 uM TnC in 10-90 |
Excite at 275 for F27W mutants, use thin black filter #51660 in the blue bag.
To Use Quinn Fluorescence to Measure Ca2+ Offrate set up for reaction is:
| Left Syringe | Right Syringe |
| 10-90 buffer plus 1mM DTT | 10-90 buffer plus 1mM DTT |
| 30 uM Ca2+ | 150uM Quinn II (2.2mM stock) |
| 6 uM TnC in 10-90 |
Excite at 330 for Quin II, use thick reflective yellow filter #57550 on side in yellow bag.
Recipe
10-90 Buffer-1 liter
- 6.7 gram KCl
- 2.1 gram MOPS
- pH to 7.0 using KOH
Bulb Replacement: Only buy 1 bulb at at time
Order from Bulb Direct, Inc
bulbdirect.com
1 Fishers Road
Pittsford, NW 14534-951
1-800-772-5267
Bulb is an OSRAM model XBO 150W/CR OFR. Cost $242 (2007).