BC-535 Preliminary, Rev.
BC-535 Preliminary, Rev. 060126 Table of Contents NOMENCLATURE.................................................................................................................................... 5 Text conventions ..................................................................................................................................... 5 Device panel abbreviations ....................................................................................................................
BC-535 Preliminary, Rev. 060126 Perfusion ............................................................................................................................................ 17 Oscilloscope ....................................................................................................................................... 17 INITIAL TEST.......................................................................................................................................... 18 Amplifier setup ...
BC-535 Preliminary, Rev. 060126 The Warner BC-535 Bilayer Clamp Amplifier is a resistive-feedback voltage clamp amplifier designed specifically for applications using planar lipid bilayer membranes. The unique circuitry and dedicated design of this amplifier allows Warner to present an instrument of broad capability and superior quality at a cost significantly below that of our competitors.
BC-535 Preliminary, Rev. 060126 NOMENCLATURE Text conventions This manual refers to amplifier controls at three functional levels; control blocks, specific controls within a block, and settings of specific controls. To reduce confusion, we have employed several text conventions which are specified below. Since our goal is to provide clarity rather than complexity, we welcome any feedback you may wish to provide. ¾ Warner Instrument product numbers are presented using bold type.
BC-535 Preliminary, Rev. 060126 CONTROL DESCRIPTION The instrument front panel is divided into six control blocks titled HOLD, OFFSET, METER, OUTPUTS, CAP COMP, and POWER. The instrument rear panel has BNC connectors for the GAIN and FILTER TELEGRAPHS, IM OUTPUT, CAP SYNC, MEMBRANE CAPACITANCE, and EXTERNAL COMMAND IN. A 9-pin DIN connector (for the headstage), a 15 pin D connector, binding posts for CIRCUIT and CHASSIS GROUND, and a SPEAKER OUTPUT are also located on the rear panel.
BC-535 Preliminary, Rev. 060126 Offset The OFFSET block contains the INPUT OFFSET and the AUTO-ZERO controls. The INPUT OFFSET section is comprised of a rotary potentiometer with low/high LED’s, the AUTO-ZERO pushbutton, the UNLOCK toggle, and an ACTIVE LED. This section is used to compensate for junction potentials produced by dissimilar solutions or other electrode potential differences. The OFFSET circuit (AUTO-ZERO and OFFSET CONTROL) must be armed prior to use.
BC-535 Preliminary, Rev. 060126 Selection of ΣVc displays the sum of all command voltages (Vm HOLD and COMMAND INPUT) applied to the headstage. The meter is capable of displaying command voltages up to ±1999 mV. The meter displays DC values and will average AC signals or pulses. Outputs The OUTPUTS block contains controls for selecting the Im GAIN of the amplifier and signal filtering using the built-in 4-pole Bessel filter. This block also contains the audio output controls.
BC-535 Preliminary, Rev. 060126 reading of the capacitance in pF. The dial is calibrated to 50 pF/turn. Power Immediately adjacent to the CAP COMP block is the master power switch for the BC-535. An LED indicates power on status. Rear panel The instrument rear panel has BNC connectors for GAIN and FILTER TELEGRAPHS, IM OUTPUT, CAP SYNC, EXTERNAL RESET IN, and EXTERNAL COMMAND IN.
BC-535 Preliminary, Rev. 060126 Note: We recommend separating the circuit and chassis grounds by disconnecting the bridging bar between the associated ground posts. Loosen the posts and slide the bridge to one side. Gain Telegraph The GAIN TELEGRAPH is a stepped voltage output designed to communicate the instrument gain setting to your acquisition software. DC voltages are stepped from 0.0 V to 5.5 V, in steps of 500 mV.
BC-535 Preliminary, Rev. 060126 Im output The Im OUTPUT signal present on the instrument front panel is mirrored on this rear panel BNC. Use of this output rather than the front panel BNC can unclutter your work environment. External Command In External COMMAND IN signals can be input via BNC connectors on either the instrument front panel or the instrument rear panel.
BC-535 Preliminary, Rev. 060126 Model membrane The BC-535 is shipped with a model membrane, the MC-1, which can be used to test the performance of the amplifier. The MC-1 contains a 100 pF capacitor connected in parallel with a 1 GΩ resistor. The precision of this resistor is ± 5%. The MC-1 connects to the two 1 mm mini-jacks on the headstage marked INPUT and REF. The green grounding wire on the MC-1 is attached to the isolated grounding jack on the headstage.
BC-535 Preliminary, Rev. 060126 SETUP For those with little experience in bilayer work, we suggest a review of Ion Channel Reconstitution edited by C. Miller, Plenum Press, New York, 1986. In particular, Chapter 5, "How To Set Up A Bilayer System", covers many important aspects of the subject. Several other pertinent references are included in the appendix at the back of this manual. Figure 1. Schematic representation of a BLM setup.
BC-535 Preliminary, Rev. 060126 grounding the Faraday cage through the headstage (see Headstage connections, page 11), we do not recommend this procedure, but instead suggest that the cage be grounded through the amplifier circuit ground. Several Faraday cage designs are available. The most common commercial design is that of a copper or aluminum wire mesh supported on an aluminum frame.
BC-535 Preliminary, Rev. 060126 solvent such as decane, is manually painted or drawn across the aperture. Excess lipids drain away from the aperture and under hydrophobic pressure the remaining lipids orient themselves into a molecular bilayer. Planar lipid bilayer membranes are routinely generated on a variety of supports including cups made from polystyrene, polysulfone, Teflon, or Delrin. Teflon sheets, Pasteur pipette tips, or plastic septa have also been used.
BC-535 Preliminary, Rev. 060126 Optionally, many researchers filter their data using an external device. These devices are normally of the low-pass 8-pole Bessel design. While Butterworth filters have steeper frequency cutoff characteristics, they are less commonly used since they tend to overshooting a rapidly varying signal thus introducing an artifact into the data.
BC-535 Preliminary, Rev. 060126 17 sufficiently little mechanical noise such that one is able to make recordings while simultaneously stirring. Warner Instruments has developed a unique stirplate specifically designed for the planar lipid bilayer. This stirplate, the SPIN-2, provides separate rotating dipoles for each side (cis and trans) of the cup/chamber and its noise-free operation allows data recording while stirring.
BC-535 Preliminary, Rev. 060126 INITIAL TEST This section describes the basic setup for incorporating the amplifier and headstage into the BLM workstation. Procedures for testing the performance of the BC-535 immediately follow. Amplifier setup The headstage connects to the amplifier via a 1.8 meter cable. It is a good idea to route the cable through a short shield (anti-wave guide) prior to its entering the Faraday cage.
BC-535 Preliminary, Rev. 060126 Set all controls on the instrument to the values specified below. Control block Control Setting HOLD OPERATE TOGGLE standby COMMANDS APPLIED TOGGLE off VOLTAGE UP/DOWN TOGGLES adjust until meter reads 0 COMMAND INPUT FRONT/REAR TOGGLE COMMAND INPUT ATTENUATION TOGGLE off x0.
BC-535 Preliminary, Rev. 060126 • Deactivate the OFFSET CONTROLS by moving the UNLOCK toggle to the unlock position. The ACTIVE LED will go off and the toggle will return to its original position. The offset reading on the METER in the METER block will not change. • Adjust the position of the trace on the oscilloscope to a convenient reference line. • Switch the SELECTOR SWITCH in the METER block to ΣVc.
BC-535 Preliminary, Rev. 060126 • Switch the STANDBY/OPERATE switch to operate. • You should observe a noise level of no greater than 6 mV p-p, equivalent to 0.171 pA RMS at this gain setting. • Place the amplifier into standby. Test instrument Im output • Switch the SELECTOR SWITCH in the METER block to Im. • Set the amplifier GAIN to 20 mV/pA • Set the oscilloscope voltage base to 0.5 V/div. • Using the UP/DOWN toggle switches in the HOLD block, adjust the Vm HOLD until it reads 40 mV.
BC-535 Preliminary, Rev. 060126 • Verify that the signal on the oscilloscope moved 1 div upwards. This corresponds to a 10 pA offset at this gain setting. • Depress the AUTO-ZERO pushbutton. • The ACTIVE LED will flash, indicating operation of the AUTOZERO function. • Verify that the Im OUTPUT signal on the oscilloscope returns to baseline. • Verify that the OFFSET reading on the METER returns to 0 mV and that the ACTIVE LED is off. • Switch the STANDBY/OPERATE switch to standby.
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.
BC-535 Preliminary, Rev. 060126 Input offset Prior to forming a bilayer membrane, the resistance of the aperture is exceedingly low, as small as 1 kΩ. Consequently, an input voltage as small as 1 mV can induce a large currents exceeding 1 nA to flow. These currents are much larger than the pA currents typical in single channel recording and can overload the headstage.
BC-535 Preliminary, Rev. 060126 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.
BC-535 Preliminary, Rev. 060126 APPENDIX Theoretical considerations Shielding Proper shielding of all cabling and recording apparatus are important in maintaining a large signal-to-noise ratio. The necessity of a high quality Faraday cage to protect the headstage from stray input signals cannot be over emphasized.
BC-535 Preliminary, Rev. 060126 connected together, or bridged. We recommend severing this connection and only using the CIRCUIT GROUND. The schemes shown below and described above indicate connection of the BC-535 CHASSIS GROUND to an external ground point. However, it is often advantageous to try different ground configurations with the BC-535 to determine which works best in your environment.
BC-535 Preliminary, Rev. 060126 Membrane capacitance calculations It is possible to theoretically derive an equation to determine the size of the bilayer formed across the aperture. While this equation will probably not yield an exact result (most likely due to variation in the dielectric constant of your lipid mixture), it will give a reasonably approximate result.
BC-535 Preliminary, Rev. 060126 where i is the current appearing at the Im output, k is a constant of proportionality, and A is the area of the forming membrane. It should be apparent from the preceding discussion that the magnitude of the current, and hence the amplitude of the resulting square wave, is linearly proportional to the area of the bilayer membrane.
BC-535 Preliminary, Rev. 060126 Specifications Noise: Measured with 8 pole Bessel filter at specified cutoff frequency Frequency range Open input 100 pF at input DC to 1 kHz 0.060 pA RMS 0.82 pA RMS DC to 100 Hz 0.009 pA RMS 0.28 pA RMS Bandwidth: 75 kHz Input commands: HOLD: Digital; 1 or 10 mV steps to ± 400 mV maximum COMMAND IN: Front and rear external input, 10 V/V (applied voltage is attenuated by 10/100/1000 at the command electrode) Junction zero: AutoZero or manual adjust.
BC-535 Preliminary, Rev. 060126 Specifications (continued) Digital meter: 3.5 digit LED ± 1999 mV full scale Junction offset: ± 120 mV full scale 0 to 1999 pF Cap Test: Vc : Im : ± 1999 mV full scale Power: ± 1999 pA full scale 100-125 or 220-240 VAC, 50/60 Hz Dimensions: H×W×D Operating Conditions: Case : 9 × 42 × 25 cm; (3.5 × 16.5 × 10 in) Headstage : 2.3 × 2.8 × 5.8 cm (0.9 × 1.1 × 2.25 in) 1.
BC-535 Preliminary, Rev. 060126 Chloriding electrodes Silver-silver chloride electrodes act as signal transducers by converting ionic currents in solution to an electric current within a wire. This is achieved by utilizing a reversible oxidation/reduction reaction between the electrode and Cl- ions in solution.
BC-535 Preliminary, Rev. 060126 Accessories and replacement parts Description Model No. Order No. Classic bilayer chamber, 1 ml working volume BCH-13A 64-0400 Classic bilayer cups, 1 ml working volume. Available in various materials and aperture sizes. 150 um aperture size shown. CD13A-150 CP13A-150 CF13A-150 64-0410 64-0404 64-0416 Classic bilayer chamber, 3 ml working volume BCH-22A 64-0401 Classic bilayer cups, 3 ml working volume. Available in various materials and aperture sizes.
BC-535 Preliminary, Rev. 060126 2. Occasionally, a knob on the front panel will loosen after long use. These are "collet" style knobs and are tightened with a screw located under the knob cap. To gain access to the adjustment screw, pry the cap off with a thin bladed screwdriver or similar tool. Warner Instruments A Harvard Apparatus Company .
BC-535 Preliminary, Rev.
BC-535 Preliminary, Rev.
BC-535 Preliminary, Rev. 060126 37 Warner Instruments A Harvard Apparatus Company .
BC-535 Preliminary, Rev. 060126 Glossary A/D converter – Analog to Digital converter. Computers are inherently digital while the voltage or current output from an amplifier is analog. Therefore, a signal must be first converted to a digitized form before a computer or its software can accept it. Desirable features in an A/D converter include rapid signal conversion, small-step resolution and low noise. analog – Continuous or non-discrete. Often dynamically varying. Compare to: digital.
BC-535 Preliminary, Rev. 060126 COMMAND IN – Also CMD IN. Command Input. An external input into the BC-535 allowing the application of user defined command voltages to the headstage. Connection is via BNC. command sensitivity – Selectable scaling of CMD IN input. Attenuation values of CMD IN are x0.1, x0.01, and x0.001. command voltage – The voltage applied to the headstage resulting in a desired transmembrane potential in the system under study.
BC-535 Preliminary, Rev. 060126 ground loop – A loop formed from multiple connections into the circuit ground plane by the same device. The flux of magnetic fields through this loop can induce small currents within the ground plane resulting in increased noise in the circuit. Careful consideration of the interconnection between several devices is often required to identify ground loops. headstage – A low gain amplifier placed as close to the preparation as possible.
BC-535 Preliminary, Rev. 060126 reset – An operation wherein the collected charge on the integrating capacitor in the headstage is dissipated, readying the system for further use. signal polarity – Defined as the sign applied to a current generated through a membrane in the presence of an applied holding potential. The electrophysiological definition is determined by the membrane such that an outward directed current and a depolarizing potential are both positive.
