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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).