PLANAR LIPID BILAYER EXPERIMENTS (1)

See also Planar (Black) Lipid Bilayer Apparatus
 

Preparation of chambers:

  1. Immediately after use, rinse with distilled water a couple of times and then with 70% or 100% ethanol (can repeat this rinsing), then invert the cells and let them air-dried.  This should work for getting rid of most porins.

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  2. When used for small peptides which can get into the porous Telfon cell wall and are hard to clean, soak overnight in RBS (diluted) at room temperature.  Then rinse under distilled water for half a day (detergent gets into cell wall too and may cause problems during experiment).  Then rinse as above.  Don’t heat the chambers (e.g. with hair-drier, that’s not good for the chamber)

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  3. Oxidized cholesterol stored at –20ºC in chloroform CHCl3 as a 2% solution in 100 µl aliquots, may be partially dried down.

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  4. Dry the aliquot down completely in a vacuum until can’t smell the CHCl3, resuspend the white powder in 0.133 µl decane with 5 µl of n-butanol to yield 1.5% solution of cholesterol, vortex, can store this in fridge, sealed to prevent moisture getting in.  Also, don’t open vial until warm (room temperature) or moisture will get into vial and hydrate the lipid.

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  5. Coat the hole in chamber with about 3 µl of oxidized cholesterol, let air-dry.  For single channel, coat one side; for selectivity, coat both sides.  Another lipid commonly used is diphytanoylphosphatidyl choline (DiphPC).  It is prepared in the same method used for oxidized cholesterol (dried down a 2% solution, resuspend in decane and n-butanol to yield the desired %).  There are small vials with 2mg/vial DiphPC in freezer.  These were originally in CHCl3 but probably all dried down after the CHCl3 evaporated.


Doing the experiment:
 

  1. Make sure the table is pumped up so the equipment will be less sensitive to motion around.

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  2. Check to ensure input voltage is ok: disconnect the blue box from the amplifier on the top and connect it to the voltmeter (while the two electrodes are in a cell with some salt solution), check to see voltage applied = what you see on voltmeter.  Change battery (2 x 9V) if it’s off.

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  3. Don’t touch calibrated knob on power box.

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  4. Set rise time to 10 ms (can give better resolution if pores are small and close together but this setting is noisier); set to 30 ms if channels are fast and fuzzy.

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  5. Set the gain and suppression, generally 109 and 10-9 respectively; can use 1010 for greater sensitivity.

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  6. Set power pack at 10-50 mV at DC for single channel measurements and selectivity measurements.  (005 = 50mV).

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  7. Oscilloscope: set at 20-50 mV per division for small pores and 100 mV or more for large pores.

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  8. Chart recorder: set at 50-200 mV full scale (span) for small pores and 1-2V for large ones.

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  9. Note on chart recorder paper: sample using (and amount used), salt solution and pH, gain, mV applied, chart speed, date, chart full scale.

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  10. Fill the chamber with salt solution (after lipid coated has been dried around the hole).  The salt solution is usually 1M KCl made up with dH2O, make sure there is no growth in solution (can filter before use).  Other solutions: 0.1M or 3M KCl, 1M LiCl, 1M Kacetate, etc. (pH 7)

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  11. Turn power pack on and check for baseline (about 20 min) to ensure there is no contamination in chamber.

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  12. Add protein of interest.  Usually 5 µl of a 1:1000 dilution of the original sample is a good place to start.  Dilute the protein in 0.1% Triton X-100 (10 µl in 10 ml total).  If using diphytanoylphosphatidyl choline, use a 1:100 or even 1:10 dilution (depend on your sample conc.)  because this lipid is less sensitive than cholesterol and need more proteins (but it also gives less artifact than with cholesterol).

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  13. Record events on chart recorder.  Watch the oscilloscope to ensure that the events on the chart recorder are real events and are single events.  Mark these events on the chart recorder.  Generally need to collect at least 100 pore events to generate a meaningful histogram.  Can also check if the pore is voltage-dependent.

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  14. If membrane breaks, reform it but do not keep adding more lipid unless you cannot reform the membrane without it.
Calculations:
V=IR
Volts = amps x ohms
Conductance 8 (Siemens) = 1/ohms = amp/volt
 # volts observed / volts applied x gain = siemen

 e.g.  20 mV size steps, 10 mV applied, 10-9 gain

20 x 10-9 mV / 10 mV  = 2 x 10-9 S = 2nS channels.


Updated 01 December 2000.