BC-535 Preliminary, Rev. 060126
23
OPERATION
The general procedure is to first set up the bilayer chamber, add solutions, and make electrical
contact. This is followed by adjusting the input offset and forming the bilayer membrane. The strategy
for incorporating channel containing membrane vesicles to the bilayer membrane will depend on the
system under study, but will normally proceed by adding vesicles or purified protein to one side of the
membrane under the appropriate ionic and/or osmotic conditions. Once a channel has been
successfully incorporated into the bilayer the solutions are perfused and initial experimental conditions
established. At this point recording of data proceeds.
Setup of the bilayer chamber
Bilayer membranes are formed across an aperture in a septum which separates two chambers.
The most common configuration is that of a cup (which supports the aperture) placed inside a holder.
The interior of the cup represents one chamber while the interior of the holder is the other chamber.
The cup wall is the septum. Electrical connections are made via agar salt bridges into each chamber.
The whole assembly must be shielded from electrical and vibrational interference to obtain low noise
recording of bilayer currents.
The aperture is prepared to accept lipids prior to membrane formation. This is achieved by
‘coating’ the hole with the lipid cocktail before adding solutions to the cup or chamber. Several
techniques are employed to coat the hole prior to membrane formation. While the choice of technique
will depend on your application, the materials at hand, and your ingenuity and training, once the hole
has been coated, the cup is inserted into the chamber and both the cup and chamber filled with the
appropriate solutions. Two methods are presented below.
One method to coat the hole uses a small (1-2 mm) ball formed on the end of a glass rod or
Pasteur pipette with a Bunsen burner. The rod is dipped into the lipid mixture and a coating of lipids is
applied to the outside rim of the hole. An advantage of this technique is that it is relatively easy to
keep the glass rod, and hence the resulting membrane, free from contamination.
An alternative method is to insert several lipid-covered hairs from a Red Sable paintbrush through
the aperture. The brush is then revolved in a small circle until the hole is uniformly coated with lipid.
(Use a size 00 or 000 Red Sable artists dotting brush which has been trimmed to present 3-5 hairs of
the same length. The brush is cleaned and dipped into the lipid cocktail before coating the hole.) As
suggested above, a disadvantage to this technique is that the brush can easily become contaminated
over time or through misuse.
The headstage leads should not be directly connected to the bathing solutions. Instead, leads are
routed to wells containing a salt solution which are in turn connected to the solution baths via agar
salt bridges. The salt bridge wells should ideally contain the same solution used in the formation of the
salt bridge, usually 1 M KCl. In addition, these wells should be adjacent to the baths so that the agar
bridges used to complete the circuit from well to bath are as short as possible. The supplied sliver-wire
electrodes require chloride-plating prior to their first use and insertion into the salt bridge wells. (See
Chloriding electrodes
, Appendix.)
Warner
Instruments
A Harvard Apparatus Company .