Planar (Black) Lipid Bilayer Apparatus
Preparation of chamber:
- To clean the chambers, soak them in 4% SDS at 70oC (preferably overnight), then ethanol at 70oC for 30 min.
- If there are still contaminating proteins in the chamber after soaking in SDS overnight – rinse the cell briefly in acetone (this dissolves the teflon cell, so don’t let it soak!).
- Dry the chamber with the hair dryer.
- The oxidized cholesterol is stored at -20oC in CHCl3 as a 2% solution in 100µl amounts in small vials (it may be partially dried down).
- Dry the oxidized cholesterol down completely in a vacuum for 2 – 4 hours (until you can’t smell the CHCl3).
- Then resuspend the white powder in 0.133 ml decane with tiny drop of isobutanol to yield a 1.5% solution of oxidized cholesterol. Vortex.
- Keep on ice but avoid getting moisture inside.
- Coat the hole in chamber with about 3 µl of oxidized cholesterol:
- For single channel measurements – coat 1 side.
- For selectivity measurements – coat both sides.
- Dry chamber with the hair dryer until the smell of decane is gone.
NB. Diphytonyl phosphatidye choline can be used in the same fashion.
Setting up machinery:
- Calibrate the oscilloscope and chart recorder with power pack.
- Set the rise time – usually 30, 10 can give better resolution if pores are small & close together but this setting is noisier.
- Set the gain and suppression – to the same setting, generally 109, 10-9 respectively – can use 1010, 1010 for greater sensitivity.
- Power pack – set at 10 to 20 mV at DC for single channel measurements and selectivity measurements.
a. don’t touch calibrated knobs!!! b. don’t leave on “store” when not using c. small pores – set for 20 to 50 mV/division d. large pores – set for up to 1 V or more
a. set at 50 to 200 mV full scale for small pores.
b. set at 1 to 2 V full scale for large pores.
c. Note on chart recorder paper:
1) pore use and amount
3) mV applied
4) chart speed
6) salt used
7) chart full scale
- Fill the chamber with salt solution (after oxidized cholesterol has been applied and dried around the hole). The salt solution is most often 1 M KCl which is made up in a volumetric flask with deionized water.
- Paint membrane over the hole in the chamber with the small plastic coated wand (turn power pack off).
- Turn power pack on and check for events to ensure that the chamber is free from contaminating pores.
- Break membrane.
- Add protein of interest. Usually 5 µl of a 1 in 10,000 dilution of the original sample is a good place to start. You are measuring single molecule events so you don’t need much! Dilute the protein in 0.1% Triton X-100 as this improves activity. Often digesting your protein sample with lysozyme prior to adding to the chamber greatly improves the signal to noise ratio and the activity.
- Reform membrane.
- 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 the beginning and end of the actual events on the chart recorder. You generally need to collect about 100 pore events to generate a meaningful histogram.
- If membrane breaks, reform it but do not keep adding more lipid unless you cannot reform the membrane without it.
V = IR thus Conductance = 1/R = I/V
i.e. Volts = amps x ohms S(siemen) = 1/ohm = amp/volt
(# volts observed/volts applied) x gain = Conductance in Siemens
example: 20 mV size steps, 10 mV applied 10-9 gain
(20 mV x 10-9/ 10) = 2 x 10-9 S = 2 nS channels
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