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.
“DISC”
“MAKE GROUP”
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..
CHEMISTRY FOR STOPPED FLOW
**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: http://www.basic.northwestern.edu/biotools/ProteinCalc.html
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
RecipeS
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
http://www.bulbdirect.com/NewSite/
1 Fishers Road Pittsford
NY, 14534-951
1-800-772-5267
Bulb is an OSRAM model: XBO 150W/CR OFR (Cost: $209.83)