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).