BC-535 Preliminary, Rev. 060126
25
To observe the formation of a membrane, set the
HOLD TOGGLE
(in the
HOLD
block) to the off
position, place the
OPERATE
/
STANDBY
switch in the operate position, and turn the
METER SELECTOR
SWITCH
to cap test. Prior to membrane formation, and if the aperture is not occluded (with a bubble,
for example), the
METER
will report 0 pF. In addition, the triangular wave generated by
CAP TEST
will
induce a trans-aperture current which exceeds the operational range of the amplifier input. This
results in the appearance of a full scale (10 V p-p) pseudo-square wave at the I
m
OUTPUT
.
During membrane formation, the initial covering of the aperture by lipid dramatically decreases
the amplitude this square wave allowing you to observe the formation of the bilayer on an
oscilloscope. Additionally, the audio signal will change character and the
METER
will begin to report an
increasing capacitance. As the bilayer continues to form you will observe a time-dependent increase in
the amplitude of the square wave on the oscilloscope representing an increase in membrane
capacitance.
In all cases, the capacitance increase is proportional to the area of the forming membrane and so
allows you to observe both the size and stability of the bilayer formed. (For additional discussion see
Membrane capacitance calculations
, Appendix.)
If you are using the brush technique, then the bilayer membrane is initially formed by painting
lipids across the hole. This is achieved by dipping a clean brush in the lipid cocktail and drawing a thin
lipid film across the open aperture (reminiscent of making bubbles when you were a kid).
Alternatively, the membrane can be formed by momentarily occluding the hole with the end of the
lipid-coated glass rod. In either case, the lipids will initially occlude the hole in a thick layer. After a
short time (several seconds to a few minutes), excess lipids will drain away from the hole until a
bilayer is formed. The area of the forming membrane can be monitored on an oscilloscope or on the
meter. In general, several attempts of the above procedure may be necessary before a stable
membrane is formed.
Once the membrane has formed and appears stable,
CAP TEST
should be turned off and the leak
conductance of the membrane checked. A good membrane will have a conductance of less than 10 pS
(i.e., 1pA/100mV).
Commands
Once a stable membrane is formed, the appropriate ionic and/or osmotic conditions are
established and channel bearing vesicles are added. The system is monitored in the presence of a
transmembrane holding potential for a vesicle ‘fusion event’. Once a channel has incorporated into the
bilayer membrane the solutions are quickly perfused to prevent further vesicle fusions and the
appropriate experimental conditions established.
Command voltages to the bilayer membrane are effected by the V
m
HOLD
control, by an externally
applied signal (
CMD IN
) or by a combination of the two. V
m
HOLD
provides a DC potential of either
polarity up to 400 mV. External signals at
CMD IN
are attenuated by x0.1, x0.01, or x0.001.
Therefore, a 10 V DC signal at
CMD IN
results in an applied voltage at the headstage of 1000, 100, or
10 mV, respectively, for attenuation settings of x0.1, x0.01, or x0.001.
Warner
Instruments
A Harvard Apparatus Company .