Archives: Methods

Reference:

Angus et al. (1982). AAC 19:299

Materials:

• Double beam P.E. spectrophotometer and chart recorder
• Small French pressure cell

Cells:

1. Grow overnight in 1 ml LB normal salt (plus marker eventually)
2. Inoculate 0.4 ml into 20 ml LB normal salt (no marker anymore, since antibiotic may interfere with the cell surface.)
3. Grow to OD600 = 0.5 -0.8 (mid-log)
4. Remove a small amount, if needed, to check cell phenotype
5. Centrifuge and resuspend so that all strains to be tested are at the same final OD (1.0) in: 10 mM Na-Hepes pH 7.0
6. 5 mM MgCl2 (to maintain OM integrity)
7. Record OD600 of resuspended cells for calculations

Break 3 ml of each cell sample 1 x in small French press, record OD600 and keep on ice until needed.

Nitrocefin (NF):

Make stock solution of 1.0 mg/ml NF by dissolving 10 mg of NF in about 200 ul DMSO. Make sure all NF has been dissolved in the DMSO before adding Hepes buffer (same as above) to make up to 10 ml. Solution should be dark yellow-coloured and clear. A cloudy solution means that the NF has come out of solution. If this occurs, you can try to save the NF by pHing it in (almost never works) or by diluting your stock solution 10-fold to make a working solution of 0.1 mg/ml. Check the pH of your Hepes solution before making another attempt.

Place 1 ml of stock solution in each of 10 Falcon 10 ml tubes, cover with aluminium foil and freeze at -20 C. Dilute in the tube with 9 ml of buffer immediately prior to use. You will need about 5-10 ml at 0.1 mg/ml pere strain for one day’s experimenting.

The diluted stock solution can be refrozen and used later if you don’t use it all. Check colour.

NOTE: NF is a suspected carcinogen, so use gloves and a mask when weighing it out and don’t spill overmuch. NF 1 x solution should be yellow in colour , not orange. If it isn’t, either the compound has been destroyed by light or your concentration is wrong. You will need to repeat broken cell assays with several different concentrations of NF.

Assay:

Whole cells

0.65 ml 0.1 mg/ml NF (for P. aeruginosa)

0.1 ml whole cells or broken cells

The concentration of NF to be used will depend on the strain and the amount of B-lactamase produced by the bacteria.

Mix briefly by upending with parafilm to seal the cuvette opening and read on strip chart recorder at OD 495.

Reference: 0.65 ml NF plus 0.1 ml cell supernatent (pellet 200 ul same cell suspension in Eppendorf for 1 min and use the supernatent to control for leakage of beta-lactamase out of the cell).

Settings:

Ordinate limits = 0-0.2 for whole cells w/o plasmid beta-lactamase; 0-0.5 for whole cells with plasmid beta-lactamase

Chart speed = 15 mm/min

Broken cells:

0.65 ml nitrocefin

0.1 ml broken cells

Same procedure as for whole cell

Settings:

Ordinate limits = 0-2.0 for determination of rate of cleavage; 0-3.0 for determination of extinction coefficient (max. OD495 after cleavage of all 0.1 mg NF in cuvette).

Objective:

To methylate fatty acids in whole cells or lipopolysaccharide.

Reagents:

• Methanol-Hydrochloride Reagent Kit
• 10mg of whole cells or 1 mg of lipopolysaccharide
• 400 nmoles of fatty acid standard (pentadecanoic acids C15)

Methods:

1. Prepare 1M MeOH-HCl reagent according to the instruction given with the kit.
2. Add internal standard (C15 fatty acid) to give a final concentration of 400 nmoles/100ml (usually 10mg of C15 in 100ml of reagent.
3. Weigh 10mg of whole cells or 1 mg of LPS into a clean screw-cap tube.
4. Add 1ml of MeOH-HCl reagent/internal standard into each tube and vortex.
5. Heat at 100^oC for 20 minutes. (Note: each tube should be very well sealed with teflon tape and grease to prevent evaporation.)
6. Sonicate and heat at 100^oC overnight.
7. Neutralize acidity with 0.5N NaOH. Test pH with pH paper.
8. Centrifuge in clinical centrifuge for 5 minutes. Save supernatent in clean glass vial.
9. Do gas chomatography with programmed REWH method.

Reference:

J. Bacteriol. 155: 831-838. (Darveau, Hancock method)

Reagents :

• 10mM Tris-HCl, pH 8.0
• 10 mM Tris-HCl, pH 8.0; 2 mM MgCl₂
• 0.5 M EDTA in 10 mM Tris-HCl, pH 8.0
• 20% SDS in 10 mM Tris-HCl, pH 8.0
• 0.375 M MgCl₂ in 95% EtOH
• 0.1 M EDTA, 2% SDS, 10 mM Tris-HCl, pH 8.0
• Deoxyribonuclease I
• Ribonuclease
• Protease/Pronase

Methods:

1. Grow 1 litre of bacterial cell in an appropriate media (Proteose Peptone No. 2 for P. aeruginosa) until an O.D. 600 nm of 0.6 – 0.8
2. Harvest cells at 7,000 rpm for 15 minutes. Lyophilize. (Note : 1 litre of wet bacterial cells equivalent to 500 mg of dried bacterial cells.)
3. Resuspend 500 mg dried bacterial cells in 15 ml of 10 mM Tris-HCl, pH 8.0, 2 mM MgCl₂.
4. Add DNase (100 ug/ml) and RNase (25 ug/ml).
5. French press the cell suspension twice at 15,000 psi.
6. Sonicate for two 30s bursts at a probe intensity of 75.
7. Add DNase and RNase to bring their final concentrations to 200 ug/ml and 50 ug/ml respectively.
8. Incubate the suspension at 37^oC for 2 hours.
9. Add 5 ml of 0.5 M EDTA(tetra sodium salt)/10 mM Tris, pH 8.0; 2.5 ml of 20% SDS/10 mM Tris, pH 8.0 and 2.5 ml of 10 mM Tris-HCl, pH 8.0 to give a final volume of 25 ml, final pH approx. 9.5.
10. Vortex and centrifuge at 50,000 g for 30 minutes at 20^oC to remove peptidoglycan.
11. Save supernatant. Add pronase to give a final concentration of 200 ug/ml.
12. Incubate at 37^oC with constant shaking overnight.
13. Add two volumes of 0.375 M MgCl₂/95% EtOH. Mix and cool to 0^oC in -20^oC refrigerator.
14. After the sample had cooled to 0^oC, centrifuge at 12,000 g for 15 minutes at 0 – 4^oC.
15. Resuspend pellet in 25 ml of 0.1 M EDTA(tetra sodium salt), 2% SDS, 10 mM Tris-HCl, pH 8.0.
16. Sonicate at a probe intensity of 75 for two 30 s bursts.
17. Incubate the solution at 85^oC for 30 minutes. Cool to room temperature. Bring pH to 9.5 by addition of 4M NaOH.
18. Add pronase to give a final concentration of 25 ug/ml. Incubate at 37^oC overnight with constant shaking.
19. Add two volumes of 0.375 M MgCl₂/95% EtOH and cool solution to 0^oC as before.
20. Centrifuge at 12,000 g for 15 minutes at 0 – 4^oC.
21. Resuspend pellet in 15 ml of 10 mM Tris-HCl, pH 8.0. Sonicate at a probe intensity of 75 for two 30 s bursts.
22. Centrifuge at 1000 rpm for 5 minutes to remove insoluble Mg/EDTA complexes.
    Step 22a. For rough LPS, wash the pellet in small volume of water; recentrifuge and add the supernatant to the outer supernatant from Step. 22.
23. Add MgCl₂ to give a final concentration of 25mM. Centrifuge at 200,000 g (45K) for two hours.
24. Resuspend pellet in distilled water. Lyophilize if necessary.
25. Do KDO assay.

CHCl₃:MeOH (2:1) Treatment (optional)

1. Add equal volume of CHCl₃:MeOH (2:1) to sample. Vortex.
2. Centrifuge at 9K for 10 minutes.
3. Discard the bottom layer (retain white precipitate).
4. Repeat the extraction (step 1 – 3).
5. Drive reminiscent CHCl₃ and MeOH by vacuum suction or lyophilize sample.

Lipopolysaccharide Of Sodium Salt

1. Dialyse the above CHCl₃:MeOH treated LPS with:
   

   0.5 mM Hepes, pH 7.4, 5 mM Na2EDTA, pH 8.0. (five times)

   5mM Hepes, pH 7.4, 50 mM NaCl. (two times)

   Distilled water (two times)

   Change dialysate every 8 hours.

2. Lyophilize sample.

Objective:

This is a test for lipopolysaccharide (LPS), an outer membrane marker. The 8 carbon saccharide 2-keto-3-deoxyoctonate is exclusively found in LPS.

Reagents:

• Periodate H₅IO₆: 2.28 g in 100 ml H₂O
• Sodium Arsenite NaAsO₂: 2.0 g in 50 ml 0.5 N HCl
• Thiobarbituric acid: 150 mg in 25 ml H₂O (warm to dissolve, make up fresh daily)
• Sulphuric acid H₂SO₄ : 0.5 N
• Butanol reagent: 5.0 ml of conc. HCl added to 95 ml n-butanol

Standard:

KDO 200 µg/ml, use 0, 10, 20, 30, 40 µl for standards. Use dH₂O to make up each sample to a total volume of 50 µl. Omit the heating Step 3 for the standards.

Method:

1. 50 µl sample
2. 50 µl 0.5 N H₂SO₄. Vortex.
3. Place in 100^oC heating block for 8 minutes. Cool to room temperature. (This step is omitted for pure KDO.)
4. 50 µl H₅IO₆. Vortex.
5. Let stand for 10 minutes at room temperature.
6. 200 µl arsenite reagent. Vortex.
7. 800 µl thiobarbituric acid reagent. Vortex.
8. Place in 100^oC heating block for 10 minutes.
9. Cool to room temperature. Add 1.5 ml butanol reagent. Vortex.
10. Centrifuge at 2,000 rpm for 5 minutes in clinical centrifuge.
11. Aspirate about 800 µl of upper butanol layer. Measure optical density at 552 and 509 nm.
12. Extinction coefficient. An OD difference OD₅₅₂ – OD₅₀₉ of 19 = 1µM KDO.

Note:

Do not rinse cuvettes with water between readings.

Pre-clean cuvettes with ethanol and dry under nitrogen.

Read samples as soon as possible. Color development is unstable.

Note in Step 3, the hydrolysis times can be changed to obtain maximal release of KDO. Eight min. should be OK for Pseudomonas and E. coli; 12-15 min. is required for more refractory LPS samples (e.g. ß-cepacia). Test this by trial and error for a new bacterium.

The b-galactosidase activity of E. coli ML-35 measured with o-nitrophenyl-b-D-galactopyranoside (ONPG) as substrate. E. coli ML-35 is a lactose, permease-deficient strain with constitutive cytoplasmic b-galactosidase activity.

References:

1. Skerlavaj, B., R. Domenico and R. Gennaro. 1990. Rapid permeabilization and inhibition of vital functions of gram-negative bacteria by bactenicins. Infect. Immun. 58: 3724-3730.
2. Lehrer, R.I., A. Barton, K.A. Daher, S.S.L. Harwig, T. Ganz, and M.E. Selsted. 1989. Interaction of human defensins with Escherichia coli. Mechanism of bactericidal activity. J. Clinical Invest. 84: 553-561.

Materials:

• ONPG stock 30 mM = 9 mg/ml in dH20
• 10 mM sodium phosphate buffer (pH 7.5) containing 100 mM NaCl. (remember: to make Na phosphate buffer…make 10 mM Na2HPO4 and 10 mM NaH2PO4 and combine until the pH is 7.5)
• E.coli strain ML-35
• Positive control; Gramicidin S, 1-5 mg/ml
• Dual beam spectrophotometer; a dual beam model is required to subtract the background activity of cells and ONPG alone.

Method:

1. Inoculate 100 mls of media ( LB or Mueller Hinton broth ) with overnight culture of ML-35.
2. Grow up cells to mid log phase ( OD 600 = 0.4-0.6 ). Spin down and resuspend in above Na phosphate buffer to OD 600 = 0.5. Don’t wash cells with extra spins. Note: cells grown to higher OD i.e. 0.9 are still OK, but use logarithmic phase cells. Spin cells at room temperature and don’t store on ice. You can use the “Silencer” centrifuge set at “5” (approx. 4000 rpm) for 10 -15 minutes to spin down cells or the Sorvall floor model .
3. Set the spectrophotometer wavelength to 405 nm.
4. Zero the spec with water or buffer.
5. Turn chart recorder on, set speed to 15 mm/min, zero chart recorder. Full scale = OD 1.
6. In order to “subtract” the background during the assay, the reference cuvette in the back will have the same amount of cells and ONPG , but using an additional amount of buffer in place of the peptide added to the other cuvette.
7. To TWO cuvettes: Add 1/20 of ONPG stock ( 30 mM = 9 mg/ml) to cell suspension to give a final concentration of ONPG of 1.5 mM. i.e. 760 ml of cells and 40 ml of ONPG.
8. To one cuvette add buffer. i.e. 8 ml. Mix and place in the reference cuvette position.(Back)
9. To the second cuvette add peptide i.e. 8 ml of (100X) stock, mix and place in the front position.
10. Monitor change in OD, usually until the OD = 1.0. Some compounds/ peptides have a long “lag” phase, so measure for 10-15 minutes if there is little or no change in OD.
11. For each sample, a new set of two cuvettes must be used; the back reference cuvette cannot just be left in the back. Do not add the ONPG to the cells until you are ready to test each sample.

Reference:

Bader, J. and M. Teuber. (1973). Binding action of Polymyxin B on bacterial membranes, to the O-antigenic lipopolysaccharide of S. typhimurium. Z. Naturforsch. 28c:422-430.

Method:

1. Prepare a standard curve (in duplicate) from a 1 mg/ml stock solution of polymyxin B using 5, 10, 20, 30, 40 and 50 ml of the stock solution and all the volumes are brought up to a final volume of 50 µl with H₂0.
2. To 50 µl of sample containing 5-50 µg of polymyxin B, add 200 µl of a 1.0% solution of Na₂B₄O₇.10H₂O in distilled H₂O and 25 µl of 100 mM 1-fluoro 2,4 dinitrobenzene in ethanol.
3. Incubate for 1 hour at 37ºC.
4. Add 1 ml of 2N HCl.
5. Add 1 ml of n-Butanol and vortex.
6. Centrifuge in a clinical centrifuge for 3 minutes at 100 rpm.
7. Read the butanol phase at OD₄₂₀.

Reference:

Schindler and Teuber. Antimicrob. Agents Chemother. 8:95-104, 1975.

Method:

1. Make column using Sephadex G-25 in a 50 x 2.5 column. (Bed vol. approx – 100 ml, uses 10-15g dry bead.) Equilibrate column with at least 100 ml of 10mM Na₂HPO₄ pH7.1/0.145M NaCl buffer.
2. Dissolve 40mgs of Polymyxin B sulfate in 1.2 ml of 0.1M NaHCO₃.
3. Dissolve 10mgs of Dansyl-chloride in 0.8 ml of acetone.
4. Add the polymyxin to the Dansyl-chloride and place in the dark for 90 min at room temperature.
5. After incubation load the mixture onto a Sephadex G-25 column (50 x 2.5 cm) equilibrated with 10mM Na-phosphate buffer (pH 7.1) containing 0.145M NaCl.
6. Collect 5-6ml fractions.
7. The Dansyl-Polymyxin comes out in a fairly broad peak ahead of the unreacted Dansyl-chloride peak. Location of the Dansyl-Polymyxin in the collected fractions can be determined by holding a UV lamp over the fractions and looking for fluorescence. The fluorescence of the Dansyl-Polymyxin is yellowish, while the unreacted Dansyl-chloride is more blue-green. The fractions containing the Dansyl-polymyxin are extracted into about 1/2 volume of n-butanol. The butanol is then evaporated to dryness in a glass petri dish place inside a dessicator which is then evacuated and placed at temperature of 37C. This takes about 24 hrs.
8. The dried Dansyl-Polymyxin is dissolved in 3 ml of buffer (5mM Hepes, pH 7.0) and stored in aliquots at -20C. The concentration of the Dansyl-polymyxin is determined by dinitrophenylation assay.

NOTE: DPX was commercially available for a few years from Molecular Probes (P13238) in addition to other fluorescently labelled polymxin B compounds, however these products have been discontinued.

1. Grow bacteria in 5ml Proteoze Peptone No2 broth (for Pseudomonas) or Luria broth (10g tryptone, 5g yeast extract, 5g Nacl per liter; for E. coli) overnight at 37ºC with appropriate antibiotics.
2. (Optional for larger prep.) Inoculate 100 ml of the corresponding broth with the overnight culture. Grow at 37^oC until an OD₆₀₀ 0.6 to 0.8 is reached (or overnight depending on application).
3. Centrifuge at 7,000 rpm for 10 minutes and resuspend the pellet in 10 mM Na₂HPO₄, 5 mM MgSO₄, pH 7.4. For E. coli, resuspend in 10 mM Tris-HCl, 5 mM MgSO₄, pH 7.4 and add Triton X-100 to a concentration of 2%, then sonicate before proceeding to step 6. NOTE: Stocks of Na₂HPO₄ and MgSO₄ solutions must be autoclaved separately.
4. Add DNase to a concentration of 50 µg/ml and French press twice at 15,000 psi. (See French Press protocol in this manual).
5. Centrifuge at 3,000 rpm for 10 minutes to remove cell debris.
6. Decant the supernatant into a 70.1 Ti tube and centrifuge at 45,000 rpm for 1 hour.
7. Resuspend the pellet (cell envelope) in distilled water.
8. For E. coli outer membranes, adjust envelope prep. in water to 10 mM Tris-HCl, 5 mM MgSO₄, pH 7.4 and add Triton X-100 to a concentration of 2%. (Be sure protein concentration is less than 10 mg/ml.)
9. Transfer to 70.1 Ti ti tube and fill tube with buffer from step 8, if necessary. Spin at 45,000 rpm for one hour in a 70.1 Ti.
10. Resuspend the pellet (E. coli outer membrane) in distilled water.
11. To enrich prep for certain porins, bring prep to 2% SDS, 10 mM Tris-HCl pH 7.4 (no EDTA). Incubate at 40ºC. for 10 min. Sonicate to get proper disruption. The protein concentration is less than 10 mg/ml. This should solubilize non-peptidoglycan associated proteins.
12. Pellet as in step 9, using buffer from step 11.
13. Repeat step 11 adding EDTA (10 mM EDTA) to the buffer (for OmpF use 0.2M NaCl also). This should solubilize peptidoglycan associated proteins.
14. Pellet as in step 9 but reduce volume, using buffer from step 13. Using the bench top ultracentrifuge (Cellulase group) and the TLA100.2 Rotor (Tufaru’s Lab) you can get the proper Xg force in less than one ml. Protein concentration should be at 10 mg/ml. SAVE supernatant.
15. Porins should be in supernatant, and the peptidoglycan in pellet.

See also Planar Lipid Bilayer Experiments

Preparation of chamber:

1. To clean the chambers, soak them in 4% SDS at 70^oC (preferably overnight), then ethanol at 70^oC for 30 min.
2. If there are still contaminating proteins in the chamber after soaking in SDS overnight – rinse the cell briefly in acetone (this dissolves the teflon cell, so don’t let it soak!).
3. Dry the chamber with the hair dryer.
4. The oxidized cholesterol is stored at -20^oC in CHCl³ as a 2% solution in 100µl amounts in small vials (it may be partially dried down).
5. Dry the oxidized cholesterol down completely in a vacuum for 2 – 4 hours (until you can’t smell the CHCl₃).
6. Then resuspend the white powder in 0.133 ml decane with tiny drop of isobutanol to yield a 1.5% solution of oxidized cholesterol. Vortex.
7. Keep on ice but avoid getting moisture inside.
8. Coat the hole in chamber with about 3 µl of oxidized cholesterol:
9. For single channel measurements – coat 1 side.
10. For selectivity measurements – coat both sides.
11. Dry chamber with the hair dryer until the smell of decane is gone.

NB. Diphytonyl phosphatidye choline can be used in the same fashion.

Setting up machinery:

1. Calibrate the oscilloscope and chart recorder with power pack.
2. Set the rise time – usually 30, 10 can give better resolution if pores are small & close together but this setting is noisier.
3. Set the gain and suppression – to the same setting, generally 10⁹, 10^-9 respectively – can use 10^10, 10¹⁰ for greater sensitivity.
4. Power pack – set at 10 to 20 mV at DC for single channel measurements and selectivity measurements.
5. Oscilloscope
   

   a. don’t touch calibrated knobs!!!

   b. don’t leave on “store” when not using

   c. small pores – set for 20 to 50 mV/division

   d. large pores – set for up to 1 V or more

   Chart recorder:

   a. set at 50 to 200 mV full scale for small pores.
   b. set at 1 to 2 V full scale for large pores.
   c. Note on chart recorder paper:
   1) pore use and amount
   2) gain
   3) mV applied
   4) chart speed
   5) date
   6) salt used
   7) chart full scale

   Doing it:

   1. Fill the chamber with salt solution (after oxidized cholesterol has been applied and dried around the hole). The salt solution is most often 1 M KCl which is made up in a volumetric flask with deionized water.
   2. Paint membrane over the hole in the chamber with the small plastic coated wand (turn power pack off).
   3. Turn power pack on and check for events to ensure that the chamber is free from contaminating pores.
   4. Break membrane.
   5. Add protein of interest. Usually 5 µl of a 1 in 10,000 dilution of the original sample is a good place to start. You are measuring single molecule events so you don’t need much! Dilute the protein in 0.1% Triton X-100 as this improves activity. Often digesting your protein sample with lysozyme prior to adding to the chamber greatly improves the signal to noise ratio and the activity.
   6. Reform membrane.
   7. Record events on chart recorder. Watch the oscilloscope to ensure that the events on the chart recorder are real events and are single events. Mark the beginning and end of the actual events on the chart recorder. You generally need to collect about 100 pore events to generate a meaningful histogram.
   8. If membrane breaks, reform it but do not keep adding more lipid unless you cannot reform the membrane without it.

Calculations:

V = IR thus Conductance = 1/R = I/V
i.e. Volts = amps x ohms S(siemen) = 1/ohm = amp/volt
(# volts observed/volts applied) x gain = Conductance in Siemens
example: 20 mV size steps, 10 mV applied 10^-9 gain
(20 mV x 10^-9/ 10) = 2 x 10^-9 S = 2 nS channels

See also Planar (Black) Lipid Bilayer Apparatus

Preparation of chambers:

1. Immediately after use, rinse with distilled water a couple of times and then with 70% or 100% ethanol (can repeat this rinsing), then invert the cells and let them air-dried. This should work for getting rid of most porins.
2. When used for small peptides which can get into the porous Telfon cell wall and are hard to clean, soak overnight in RBS (diluted) at room temperature. Then rinse under distilled water for half a day (detergent gets into cell wall too and may cause problems during experiment). Then rinse as above. Don’t heat the chambers (e.g. with hair-drier, that’s not good for the chamber)
3. Oxidized cholesterol stored at –20ºC in chloroform CHCl₃ as a 2% solution in 100 µl aliquots, may be partially dried down.
4. Dry the aliquot down completely in a vacuum until can’t smell the CHCl₃, resuspend the white powder in 0.133 µl decane with 5 µl of n-butanol to yield 1.5% solution of cholesterol, vortex, can store this in fridge, sealed to prevent moisture getting in. Also, don’t open vial until warm (room temperature) or moisture will get into vial and hydrate the lipid.
5. Coat the hole in chamber with about 3 µl of oxidized cholesterol, let air-dry. For single channel, coat one side; for selectivity, coat both sides. Another lipid commonly used is diphytanoylphosphatidyl choline (DiphPC). It is prepared in the same method used for oxidized cholesterol (dried down a 2% solution, resuspend in decane and n-butanol to yield the desired %). There are small vials with 2mg/vial DiphPC in freezer. These were originally in CHCl₃ but probably all dried down after the CHCl₃ evaporated.

Doing the experiment:

1. Make sure the table is pumped up so the equipment will be less sensitive to motion around.
2. Check to ensure input voltage is ok: disconnect the blue box from the amplifier on the top and connect it to the voltmeter (while the two electrodes are in a cell with some salt solution), check to see voltage applied = what you see on voltmeter. Change battery (2 x 9V) if it’s off.
3. Don’t touch calibrated knob on power box.
4. Set rise time to 10 ms (can give better resolution if pores are small and close together but this setting is noisier); set to 30 ms if channels are fast and fuzzy.
5. Set the gain and suppression, generally 10⁹ and 10^-9 respectively; can use 10¹⁰ for greater sensitivity.
6. Set power pack at 10-50 mV at DC for single channel measurements and selectivity measurements. (005 = 50mV).
7. Oscilloscope: set at 20-50 mV per division for small pores and 100 mV or more for large pores.
8. Chart recorder: set at 50-200 mV full scale (span) for small pores and 1-2V for large ones.
9. Note on chart recorder paper: sample using (and amount used), salt solution and pH, gain, mV applied, chart speed, date, chart full scale.
10. Fill the chamber with salt solution (after lipid coated has been dried around the hole). The salt solution is usually 1M KCl made up with dH2O, make sure there is no growth in solution (can filter before use). Other solutions: 0.1M or 3M KCl, 1M LiCl, 1M Kacetate, etc. (pH 7)
11. Turn power pack on and check for baseline (about 20 min) to ensure there is no contamination in chamber.
12. Add protein of interest. Usually 5 µl of a 1:1000 dilution of the original sample is a good place to start. Dilute the protein in 0.1% Triton X-100 (10 µl in 10 ml total). If using diphytanoylphosphatidyl choline, use a 1:100 or even 1:10 dilution (depend on your sample conc.) because this lipid is less sensitive than cholesterol and need more proteins (but it also gives less artifact than with cholesterol).
13. Record events on chart recorder. Watch the oscilloscope to ensure that the events on the chart recorder are real events and are single events. Mark these events on the chart recorder. Generally need to collect at least 100 pore events to generate a meaningful histogram. Can also check if the pore is voltage-dependent.
14. If membrane breaks, reform it but do not keep adding more lipid unless you cannot reform the membrane without it.

Calculations:

V=IR
Volts = amps x ohms
Conductance 8 (Siemens) = 1/ohms = amp/volt
# volts observed / volts applied x gain = siemen e.g. 20 mV size steps, 10 mV applied, 10^-9 gain

20 x 10^-9 mV / 10 mV = 2 x 10^-9 S = 2nS channels.