Biology Reference
In-Depth Information
and small changes in the bath level that are almost inevitable during perfusion
changes will change the amplitude of the capacitative current. This can produce a
shift of the instantaneous current-voltage relationship along the current axis,
which can easily be misinterpreted as a shift in the reversal potential of Icrac.
IV. Materials
Although all patch clampers have their favorite vendors, as government-
employed scientists, we cannot reveal our specific preferences here (see
Table IV
). If there is someone else at your institution who is patch clamping
successfully, you should consult them first anyway and consider buying what
they have after you try it because they will already be familiar with its operation.
V. Discussion
The patch clamp methods described here are not easy. They have daunting
initial costs in instrumentation, almost $100,000 at current list prices, and a steep
learning curve for both the technical aspects and conceptual foundations of
electrophysiology. Even for experienced investigators, the patch clamp technique
is a laborious method that requires constant trouble shooting. Nevertheless, like
any physical activity, patch clamp performance is always enhanced mysteriously
by a belief in success. Thus, a common refrain around our laboratory is ''If Neher
or Sakmann could do it, then you can do it too.''
Table IV
Components of a patch clamp rig
Tungsten wire for sylgarding and polishing
Silver wire and pellets
Pipette glass
Pipette puller
Dissecting microscope on swing arm for sylgard coating
Pipette polisher with
Inverted microscope with
LWD objective
Coarse manipulator for adjusting heating element
Fluorescent microscope with di
V
erential interference contrast optics
LED illumination is cheaper, cooler, and electrically quieter
3D micromanipulator with submicron resolution and no drift
Patch clamp amplifier, computer interface, and software
Computer according to the software manufacturer's specifications
Physiological chamber and perfusion apparatus
Pipette holder and agar bridge ground electrode
Small cylindrical sonicator
40
