The methods used for black lipid bilayer experiments have been described previously in detail (Benz et al. 1978 (link), 1979 (link)). The instrumentation consisted of a Teflon chamber with two water-filled compartments separated by a thin wall and connected by a small circular hole with an area of 0.4 mm2. The membranes were formed from a 1% (w/v) solution of diphytanoyl phosphatidylcholine (DiPh-PC) (Avanti Polar Lipids, Alabaster, AL) in n-decane by painting onto the hole a 1% (w/v) solution of the lipid in n-decane. The protein-containing fractions were added to the aqueous phase after the membrane had turned black to one side of the membrane (the cis-side). The membrane current was measured with a pair of Ag/AgCl electrodes with salt bridges switched in series with a voltage source and a highly sensitive current amplifier (Keithley 427, Cleveland, Ohio). The temperature was kept at 20 °C throughout. Selectivity measurements were performed by establishing a fivefold KCl-gradient (0.1 M versus 0.5 M KCl) across lipid bilayer membranes containing about 100–1000 pores formed either by PorARc, PorARr, and PorBRr alone or by a combination of PorARr and PorBRr. The zero-current membrane potentials were measured with a Keithley 617 electrometer and analyzed using the Goldman–Hodgkin–Katz equation (Benz et al. 1979 (link)).
Diphpc
Manufactured by Avanti Polar Lipids
Sourced in United States
DiPhPC is a synthetic phospholipid used in biochemical and biophysical research. It serves as a structural component in the formation of model membranes and liposomes. DiPhPC exhibits a neutral charge and can be used to study the behavior and properties of lipid bilayers.
Automatically generated - may contain errors
Lab products found in correlation
Clampfit software, by Molecular Devices (2 mentions)
Originpro 8, by OriginLab (1 mentions)
Keithley 427, by Tektronix (1 mentions)
Asolectin, by Merck Group (1 mentions)
Originpro 2021, by OriginLab (1 mentions)
Derlin cuvette, by Warner Instruments (1 mentions)
Bilayer clamp amplifier, by Warner Instruments (1 mentions)
Hexadecane, by Merck Group (1 mentions)
Cholesterol, by Merck Group (1 mentions)
Gdnhcl, by Merck Group (1 mentions)
7 protocols using diphpc
1
Characterizing Membrane Pore Formation
2
Phospholipid Bilayer Formation and Ion Channel Characterization
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The painting method was used to form phospholipid bilayer using 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DiPhPC, Avanti Polar Lipids). Bilayer was formed at the 50 -100 µm diameters apertures of Delrin cuvettes as previously described 14 (link) . Briefly, the aperture was pretreated with 25 mg/mL of DiPhPC in decane and allowed to dry. Bilayers were formed using the painting method after filling up the cuvettes with the recording solution (150 mM KCl, 20 mM HEPES, pH 7.4) on both sides of the chamber. Ion currents were measured using standard silver-silver chloride electrodes from WPI (World Precision Instruments) that were placed in each side of the cuvette. Measurements of the conductance of single channels were performed by painting the protein to the cis side of the chamber (the side connected to the ground electrode). Spontaneous channel insertion was typically obtained under an applied voltage of 20 mV. Conductance measurements were performed using an eONE amplifier (Elements) with a sampling rate of 10 kHz (809.1 µs interval). Traces were filtered by low-pass Bessel filter at 10 Hz for analyses performed with Origin Pro 8 (OriginLab) and Clampfit software (Molecular devices).
3
Lipid Membrane Insertion and Pore Formation
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Membrane insertion and pore activity were measured using black lipid membranes composed of diphytanoyl phosphatidylcholine (DiPhPC). Black lipid bilayer experiments were performed as described previously (3 (link)). The instrumentation consisted of a Teflon chamber divided into two compartments by a thin wall and connected by a small circular hole (∼0.4 mm2). The aqueous solutions of both chambers contained 1 M KCl and were buffered with 20 mM tris-HCl (pH 7.4). After painting a 1% solution of DiPhPC (Avanti Polar Lipids, Alabaster, AL) dissolved in n-decane across the hole to form a membrane, the toxins (3.5 nM) were added to one side of the membrane (the cis side). After 5 min, by the addition of 1.85% HCl into the same compartment containing the toxins, the pH drop to pH 4.8 was obtained. The current of the membrane was measured by using Ag/AgCl electrodes (with salt bridges) switched in series with a voltage source and a homemade current amplifier. The applied membrane potential was −50 mV at room temperature. The signal was recorded on a strip chart recorder (Rikadenki, Freiburg, Germany).
4
Planar Lipid Bilayer Technique for Toxin Analysis
5
Phospholipid Bilayer Formation and Ion Channel Measurement
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The painting method was used to form phospholipid bilayer using 1,2-diphytanoyl-sn-glycerol-3-phosphocholine (DiPhPC, Avanti Polar Lipids). Bilayer was formed at the 50–100 µm diameters apertures of Delrin cuvettes as previously described20 (link). Briefly, the aperture was pre-treated with 25 mg/mL of DiPhPC in decane and allowed to dry. Bilayers were formed using the painting method after filling up the cuvettes with the recording solution (150 mM KCl, 20 mM HEPES, pH 7.4) on both sides of the chamber. Ion currents were measured using standard Ag-AgCl electrodes from WPI (World Precision Instruments) that were placed in each side of the cuvette. Measurements of the conductance of single channels were performed by painting the protein to the cis side of the chamber (the side connected to the ground electrode). Spontaneous channel insertion was typically obtained under an applied voltage of 20 mV. Conductance measurements were performed using an eONE amplifier (Elements) with a sampling rate of 10 kHz (809.1 µs interval). Traces were filtered by low-pass Bessel filter at 10 Hz for analyses performed with Origin Pro 2021 (OriginLab Corp.) and Clampfit software (Molecular devices).
6
Planar Lipid Bilayer Experiments with mVDAC3
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Planar lipid bilayer experiments were performed as described previously [62 (link),63 (link)]. Artificial membranes made of 1% (w/v) diphytanoyl phosphatidylcholine (DiphPC) (Avanti Polar Lipids, Alabaster, AL, USA) in n-decane were painted on a 200 µm hole in a Derlin cuvette (Warner Instruments, Hamden, CT, USA). All the experiments were carried out at RT. Membrane capacitances of 100–150 pF were established for appropriate lipid bilayers. Mutant or native mVDAC3 were added from the protein stock solution of 1 mg/mL to the cis side of the cuvette filled with symmetrical 1 M KCl/10 mM HEPES pH 7.0. The single channel conductance of the pores was measured upon application of a fixed membrane potential (+10 mV) [58 (link)]. The voltage dependence was calculated by applying a triangular voltage ramp from 0 to ±50 mV of 100 ms duration, with a frequency of 10 mHz. At least three independent experiments were performed for each protein. A Bilayer Clamp amplifier (Warner Instruments) at 100 ms/point and filtered at 300 Hz was used for data acquisition. Analyses were performed with the pClamp software (Ver-10; Molecular Devices, San Jose, CA, USA).
7
Purification and Preparation of Biomolecules
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Ultrapure chemicals including LDAO, GdnHCl (guanidine hydrochloride), hexadecane and cholesterol were procured from Sigma-Aldrich Co. LLC. DDM (n-dodecyl β-D-maltopyranoside) and DiPhPC (1,2-diphytanoyl 3-phosphocholine) were purchased from Avanti Polar Lipids, Inc. Urea solutions were deionized and used immediately.
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