Calcium-Binding Protein Protocols Calcium-Binding Protein Protocols

Flow Dialysis 11
11. Similarly, add 5 to 10 µL of the unlabeled Ca 2+ successively at every six tubes
collected. The recommended total amount of Ca 2+ to be added at each step of the
successive six steps is 2/10 of the total number of the Ca 2+ -binding site, which
covers to 1.4 times the total Ca 2+ -binding sites.
12. Finally, add 10 µL of 1 M CaCl 2 to chase practically all of the bound 45 Ca 2+ , and
collect six more tubes, then switch off the pump and the fraction collector. Connect
the effluent tip to the bottle for the waste 45 Ca 2+ .
13. Take out a constant volume of the effluent in each collected tube and quantify
45 Ca 2+ with the liquid scintillation counter. Make a titration curve to confirm that
steady-state has been reached at each titration step as shown in Fig. 4A.
3.3. Disassembly of the Flow-Dialysis Cell
1. Start the peristaltic pump to wash out the radioactive solvent in the lower chamber
with the fresh solvent. Collect the radioactive effluent into the bottle for waste
45 Ca 2+ . Then switch off the pump.
2. Take out the radioactive sample solution in the upper chamber and transfer into
the bottle for waste 45 Ca 2+ with use of a Pasteur pipet.
3. Add a small amount of the detergent solution into the sample chamber, rinse with
it the inner surface and transfer the resulting radioactive solution into the bottle
for waste 45 Ca 2+ . Repeat at least three times to remove the radioactivity.
4. Take out the dialysis cell from the incubator, place it on the bench, and discharge
the solvent in the lower chamber into the bottle for waste 45 Ca 2+ .
5. Disassemble the apparatus carefully with releasing screws. With tweezers, put
the upper and lower chambers and stirring bars into the detergent solution, and
the dialysis membrane into the can for the radioactive waste.
6. Wash the disassembled parts thoroughly with detergent solution, rinse with the
distilled water.
3.4. Calculation to Make a Ca2+ -Binding Curve
1. Average the steady-state values of the radioactivity usually obtained in two or
three tubes just prior to the addition of next Ca2+ and subtract the averaged
baseline value obtained before the initial addition of 45Ca2+ . Then, correct for the
dilution at each step of titration to yield the net average value of radioactivity, C1, C2,….., Cn at each step of titration.
2. Considering the dilution factor again, calculate the total concentration of Ca2+ ;
Ca1, Ca2,……., Can at each step from the amounts of added Ca2+ and initial concentration
of Ca2+ in the protein solution determined by atomic absorption spectrometry.
3. Calculate the concentration of free Ca2+ in the upper chamber at each step from
[Ca2+ ] free = CaiCi/C n.
4. Calculate concentration of bound Ca2+ from the difference between concentrations
of total Ca2+ and free Ca2+ at each step, which gives a molar ratio of bound
Ca2+ to the Ca2+ -binding protein (Ca2+ -binding number) considering the dilution
factor in the calculation of the concentration of Ca2+ -binding protein.