SDx Protocol 1

Membrane Formation:-

Membrane formation

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

Ensure all buffer solutions are at room temperature before addition (to avoid thermal shock on the membrane) Start with 0mV bias and obtain baseline

• Remove 200uL buffer and immediately replace with 200uL compound at lowest

concentration. Allow to sit on the membrane for at least 5 minutes or until conduction plateaus** Repeat from * to **, until the highest concentrations are read or the membrane ruptures. Record time of addition for each of the above and from this calculate Gm vs concentration