Difference between revisions of "SDx Protocol 1"

Revision as of 18:13, 7 August 2013

Membrane formation

Assemble the tethaPlate. To each well add 8-10 µL AM199 lipid mixture, and incubate for 2 minutes. Now add 200 µL PBS per well followed immediately by three 200 µL PBS rinses.

Stock solution

Make a 1 mM stock solution of the ionophore in methanol. Using the method of serial dilution prepare a series of solutions in decade concentration increments, allowing 200 uL per test well so that you have solutions of 1, 10, 100, 1000, 10000 nM. The 10000 nM solution will contain 1% methanol which still means that the solution is mainly water. Higher methanol concentrations can disrupt the membrane, or allow incomplete partinioning of the ionophore into the lipophillic membrane.

If you need to use the methanol stock solution to make more concentrated test solutions then you will need to make a more concentrated stock solution (eg 10 or even 100 mM).

Compound Addition

Ensure all solutions are thermally equilibrated at room temperature before starting.

Start recording with the tethaPlate fitted to the tethaPod unit, precees for several minutes until stable baseline signals are obtained for each of the sample wells.

1. Withdraw 200 µL of the PBS buffer from the tethaPlate well.
2. Immediately replace with 200 µL test solution at lowest concentration.
3. Record the membrane conductivity for at least 5 minutes, or until the signal plateaus.
4. Withdraw 200 µL of the old test solution from the tethaPlate well.
5. Immediately replace with 200 µL test solution at the next lowest concentration.
6. Record the membrane conductivity for at least 5 minutes, or until the signal plateaus.
7. Repeat from * to **, until the highest concentrations ir reached (or until the membrane ruptures)

Record time of addition for each of the above and from this calculate Gm vs concentration