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Comparative Physiology @ Guelph

Set-up for Stopped Flow

Set Water bath temp first


Turn on stopped flow machine in following order


1) Power supply for lamp

2) Lamp trigger – green button

3) Monochomotor

4) Piston Housing

5) Channel Box

6) CPU

7) Monitor


Turn on Gas make sure set to 130 PSI


Ensure that the “earthing spring” (see page 10 manual) is in direct contact with the shaft of the plunger.  If it is not charge will begin to build up during successive runs and injections will become inhibited.


Initiate soft ware


In Main menu highlight “Newdata”

Change absorbance to emission mode in “Detector “ box

Highlight “over sample” box

Change “Internal trigger” to “external trigger”

Set excitation wave length by using middle button of mouse to highlight up arrow then type in 330 for quin or 275 for F27W mutants


In Main Menu pick “Display” than select “Overlay”


Pick “Autoscale” and “Show fit”


Set observable volts to a little over 4 by using mouse

 Setting up “Group” on hard-disc for data storage.   



Use date for Group name hit enter


Running and Fitting a protein

 When starting a new protein let 3 injections go through the machine to rinse out any water from washing NEWDATASet time (20 –200 ms depending on protein)Set number or runs “Acquire”    When finished with a protein, wash out lines by pumping water through machine.  Use plastic syringe to fill injection syringes with water..

**note always use same (left) syringe housing for Protein


Use ABS at 280 with appropriate extinction coefficient to calculate protein concentration

To calculate extinction coefficient:

Protein                                                 Ext Coefficient


WT rabbit sTnC                                  2680

WT rat cTnC                                       4080

F27W rabbit sTnC                              8370

F27W rabbit cTnC                              9770

Whole cTn                                           36040


For example if OD280 for F27W cTnC is 1.8062, % by extinction coefficient (9770)

To calculate M conc 1.8062 % 9770 = .000185 M  = 185 uM  To use tryptophan fluorescence to measure Ca2+ off rate set up for reaction is as follows 

            Left syringe                                                    Right syringe

              buffer                                                              buffer            

               200 uM Ca2+                                                   10 mM EGTA

               6 uM TnC in buffer 

Excite @ 275 for F27W mutants use thin black filter #51660 in blue bag


To use Quinn fluorescence to measure Ca2+ off rate set up for reaction is as follows 

Left syringe                                                    Right syringe


            buffer                                                              buffer

            30 uM Ca2+                                                     150 uM Quinn II (2.2 mM stock)

6 uM TnC in buffer

 Excite @ 330 for Quin II use thick reflective yellow filter that says 57550 on side in yellow bag


 20-250 buffer 

20 mM MOPS

250 mM KCl

5 mM MgCl2

1 mM DTT


pH to 7 using KOH


Quin 2 (~2 mM)

 Purchase Quin-2, in 50 mg quantities, from Calbiochem  Cat# 551826.  Order only as needed

 Calculate the concentration of the Quin-2 using its extinction coefficient, 4000 at l = 353 nM

 Dissolve Quin 2 in appropriate volume of 20mM MOPS, pH 7

 For example 50 mg of Quin 2 gets dissolved in 32.8 ml of 20mM MOPS

 To calculate concentration, dilute a sample by a factor of 10 (150 Quin 2 + 1350 buffer) then read at l=353.  Abs should be around 0.7-0.8. Divide Abs by 4000 then multiply by 10 (dilution factor) to get M concentration.

 Aliquot out Quin 2 in volumes (~400 ul) required to make up 5000 ul of the Quin 2 reaction buffer based on calculated concentration.  Place in small cardboard box, wrap box in foil and freeze.

 Thaw slowly on ice when needed.

 Before using new batch of Quin 2 test by running it against buffer plus 30 uM of Ca without protein. Set time to 20 sec.  If Quin is good will get a flat line as it should bind Ca so fast thaqt you can’t see the rate.  

Bulb Replacement  (only buy 1 at a time) Contact UW Scientific Instruments and they will install (instructions are in the hardware manual)


Order From:

Bulb Direct, Inc

1 Fishers Road Pittsford

NY, 14534-951



Bulb is an OSRAM model: XBO 150W/CR OFR (Cost: $209.83)