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Comparative Physiology @ Guelph

Complexing whole Tn




Weigh out, dissolve and measure concentration of TnI, TnC, TnT

Mix proteins in the following proportions I:C:T  1.16:1.0:1.72

Dialyze over 4 days through series of 6 buffers

Centrifuge to remove components that have come out of solution

Measure protein concentration

Concentrate using spin vials if necessary.


Step by Step


(using an actual complexing as example)


Weigh out:      35 mg TnT

                        25 mg TnI

                        TnC already in solution (7.19 mg/ml)


Dissolve TnT in 3.5 ml Hydration Buffer

Dissolve TnI in 2.5 ml Hydration Buffer


Stir separately in 2 small (XX ml) beakers until dissolved


Take 100 ul of each dissolved protein and dilute with 900 ul of Hydration buffer (10 fold dilution)


Measure concentration using a spectrophotometer scan (wave length = 250 to 350nm)

Take peak of each protein around 276 nm.  After scan made add 1000 ul sample back into beaker of the respective dissolved protein.


Divide value obtained for TnI by 0.42

Divide value obtained for TnT by 0.52

Divide value obtained for TnC by 0.2


This gives concentration in mg/ml




For this example absorbance of TnI was 0.413 therefore divide by 0.42 then multiply by 10 (dilution correction)


= 9.83


Now need to correct for adding in 900 ul buffer after doing scan


=(9.83 x 2.5)/ 3.4                        (3.4 = 2.5 + 0.9)

=7.22 mg/ml

As have 3.4 ml there fore have a total of 24.58 mg of TnI


Same calculation for TnT  (using 0.52) gives concentration of 9.98 mg/ml with total = 43.91 mg.


TnC = 7.19 mg/ml


Need to mix together proportions I:C:T =  1.16:1.0:1.72

This ratio required: 3.4 ml TnI: 2.95 ml TnC: 3.64 ml TnT


In this particular instance TnI was the limiting ingredient due to its concentration.  Therefore its volume/concentration was calculated to be equal to 1 and the proportions of the other components were adjusted accordingly.  For example to determine the proportion of TnC with TnI equal to 24.58 mg means that we need to add in 21.18 mg of TnC (see below for calculation) this is equal to 2.95 ml (21.18 mg / 7.19 mg/ml) of TnC stock.


1/1.16 = 0.86


24.58 x 0.86 = 21.18 mg TnC

 To mix:

In small beaker have TnI mixing slowly then add TnC drop by drop

When finished allow to mix for 5 min

Add in TnT drop by drop

Mix 30 min

Add in CaCl2 stock to 1.25 mM

Mix 30 min


Transfer entire volume to dialysis tubing and place into Dialysis Buffer 1



Schedule for Dialysis (Do in Cold Room)


Dialysis Buffer           Time of Day               Volume           Stir/no stir

Buffer 1                      Overnight                    250 ml             no stir

Buffer 2                      Next Day                    250 ml             no stir

Buffer 3                      Overnight                    250 ml             no stir

Buffer 4                      Next Day                    250 ml             no stir

Buffer 5                      Overnight                    250 ml             no stir

Buffer 6                      Next Day                    500 ml             no stir

Buffer 7                      Overnight                    1000 ml           stir


Total = 3 days and 4 nights


When dialysis finished, divide sample up into 1.5 ml epindorph tubes and centrifuge in small bench to centrifuge (1 speed) for 10 min.  Pipette off the supernatant from all tubes into 1, 15 ml tube.  Now do an absorbance scan on sample to determine concentration.  Require around 60 uM or 5mg/ml.  If not this concentrated; concentrate using spin kit (get from Charles).  Store on ice until used for off rate measurements.


BUFFERS TO MAKE (pH at room temperature)


Hydration Buffer (make 250 ml then freeze in 15 ml aliquots)

6 M Urea

1M KCl

25 mM Tris


pH to 8.0 using HCl.

 Dialysis Buffer 1 (250 ml)

4 M Urea

1M KCl

20 mM MOPS

5 mM MgCl2

1 mM CaCl2

pH to 7.0 using KOH

 Dialysis Buffer 2 (250 ml)

3 M Urea

1M KCl

20 mM MOPS

5 mM MgCl2

1 mM CaCl2

pH to 7.0 using KOH

 Dialysis Buffer 3 (250 ml)

2 M Urea

0.75 M KCl                            *****note change in KCl*****

20 mM MOPS

5 mM MgCl2

1 mM CaCl2

pH to 7.0 using KOH

Dialysis Buffer 4 (250 ml)

1 M Urea

0.75 M KCl

20 mM MOPS

5 mM MgCl2

1 mM CaCl2

pH to 7.0 using KOH

Dialysis Buffer 5 (250 ml)

0 M Urea

0.75 M KCl

20 mM MOPS                                     *****note lack of CaCl2 and Urea*****

5 mM MgCl2

pH to 7.0 using KOH

 Dialysis Buffer 6  (500 ml)

0 M Urea

0. 50 M KCl                                                   *****note change in KCl*****

20 mM MOPS                                    

5 mM MgCl2

pH to 7.0 using KOH

 Dialysis Buffer 7  (1000 ml)

0 M Urea

0. 250 M KCl             *****note change in KCl and addition of DTT and BME*****

20 mM MOPS                                    

5 mM MgCl2

1 mM DTT

15 mM BME

pH to 7.0 using KOH