Table 1 ∣.

Troubleshooting table

Step	Problem	Possible reason	Solution
‘Equipment setup’, ‘Preparation of the observation chamber’	PDMS support is not properly cured	Wrong curing time	Find the optimal PDMS curing time by repeating the procedure several times. As an alternative to a PDMS-made support, small ribbons of biocompatible double-sided adhesives can be used (such as the GraceBio SecureSeal Adhesive). However, chamber contamination by the adhesives during membrane painting can occur
‘Equipment setup’, ‘Electrical noise check’	Excessive electrical noise	Improper shielding and/or grounding	Check that you have connected all the metallic parts of the setup to grounding point(s); avoid ‘ground loops’; ensure that all the equipment’s ground outlets are connected to a single grounding point
6	Thick lipid films fail to spontaneously convert to lipid bilayers	Excessive amount of lipids on the grid mesh	Clean the brush (Step 3); use the clean brush to remove excess lipid material
	Lipid bilayers break spontaneously during the process of/shortly after painting	Impurities are triggering nucleation of pores and membrane rupture	Verify that lipid reservoirs (Fig. 2b) are not in contact with the PDMS support. Increase the lipid concentration in squalene. If nothing above works, change your lipids and/or solvent
15	Bad gigaseal. $G_{seal}$ (Fig. 1b) is >100 pS and/or unstable	Contaminated pipette tip	Use freshly made pipettes and store pipettes in a closed jar to protect the tips from dust. Fire-polish the pipette tip right before use. Verify that a constant positive pressure is applied to the pipette during manipulations in solution; constant efflux for the tip diminishes the tip contamination risk
		Inappropriate type of glass used	Change the type of the glass capillary you use (see ‘Equipment setup‘, ‘Patch-clamp and delivery pipettes’); if conventional patch-clamp and borosilicate capillary fail, consider using a thick-walled capillary or quartz
18	The measuring current does not increase with the pipette movement toward the membrane reservoir	The NT is broken or its lumen is inaccessible or clogged	Remove and discard the patch-clamp pipette. Repeat one to three times from Step 7; an NT should form in 50–90% of the attempts Decrease the deviation of the pipette axis from the vertical Decrease the speed of the pipette movement. Consider raising the pipette incrementally, in small steps See Troubleshooting advice for Step 6
19A(iii)	The dependence of $I_{\mathrm{m}}$ on changes of the NT length is not hyperbolic; the data fitting (Box 1) is not reliable	Nano-positioner is not calibrated	Label the tip of a patch-clamp pipette with a fluorescent marker (e.g., soak the pipette in a solution of 100-nm fluorescent microspheres). Using this pipette as a marker, make a calibration curve relating the position of the tip (in the vertical direction) and the reading of the nanoposition controller. Verify that the curve is reproducible in repetitive raising/lowering of the pipette
		The NT conductance is unstable at constant length (drift)	See Troubleshooting advice for Step 19B(i)
19B(i)	Conductance changes at a fixed position of the nanopositioner	Patch-clamp pipette and/or grid mesh move slowly	Using phase-contrast optics (40× or 60× objective) determine whether the pipette and/or grid move. Identify and eliminate the reason of the drift (loose attachment of the grid/pipette, rotation of the pipette holder, mechanical drift of the micromanipulator and/or microscope stage)
19C(i)	$I∕U$ characteristic is either linear or not symmetric	Wrong voltage range used	Increase $U$. Chose the nanotube with high $r_{\text{NT}}$ value (low tension) $I∕U$ asymmetry is seen with charged membrane due to electrophoresis in the inner lipid monolayer. Reduce $U$, apply fast voltage ramps
19C(iii)	$k$ is highly variable; the distribution of the $k$ values is not a sharp Gaussian	Poor membrane material	Change lipid stocks
20	Low SNR	Vibrations of the patch-clamp pipette	Check that your setup is properly protected against vibrations
25	Displacements of the delivery pipette cause acute and irreproducible changes in the NT conductance	Mechanical interference or large efflux from the delivery pipette	Check that the delivery pipette is mechanically isolated from the patch-clamp pipette (look for, e.g., entangled cords, physical contact between the micromanipulators; verify that the delivery pipette’s controller is not causing vibrations). Reduce the approach speed. Trace the efflux, for example, by adding fluorescently labeled nanospheres to the delivery pipette; verify that the spheres escape the pipette slowly
27	Upward stepwise changes of $I_{\mathrm{m}}$	Membrane poration	Change the osmoticant. If the problem persists, it indicates that the poration is caused by the curvature stress; the effect depends on the lipid composition used. Change the lipid composition or avoid high NT constriction
	Acute drop of the NT conductance to the background level	NT scission	Avoid extreme NT constrictions causing scission
35	No NT constriction	Delivery pipette is clogged	Change the delivery pipette, using a low-magnification microscope verify that the pipette tip is clean
	Gradual conductance decrease; no steps are observed	Low SNR	Decrease the NT length. Verify the SNR curve (Step 20). Use NTs with higher $r_{\text{NT}}$ values (lower lateral tension). To further increase SNR high-ionic-strength (1 M) solution can be used in the patch-clamp pipette
	Upward stepwise changes of $I_{\mathrm{m}}$	Membrane poration by the protein	Use dialysis to remove detergent from the protein solution. Decrease the protein concentration. To further assess the effect, the protein can be added to the patch-clamp pipette at Step 7. Continue the protocol until Step 15; do not rupture the patch. Apply the main acquisition protocol (Step 19A(i)), monitor the patch conductance, and look for upward channel-like events. The channel-like activity, if detected, can be minimized by optimizing lipid composition, for example, by adding cholesterol or lipids promoting inverted hexagonal phase appearance
48	The usNT radius variations from membrane to membrane are small	Membranes on the different holes of the grid mesh share the same lipid reservoir	Prepare another grid and repeat the protocol