إعداد طبقات ثنائية الاصطناعي للتجارب الكهربية

Hello, I'm Dr.Olaf Anderson in the Department of Physiology and Biophysics at Weill Cornell Medical College in New York City. Hello, I'm Ruchi Kaur, an MD PhD student in Dr.Olaf Anderson's lab. Hello, my name is John Kim.

I'm a research technician. Today we will show you how to prepare for single channel experiments in plain lipid bilayers. We'll show you how to prepare the chamber and we'll show you how to prepare the Bilayer Punch electrodes.

We used to isolate the small areas of membrane where we do the measurements. So Let's get started. Our electrophysiological setup consists of the following, an air suspended anti vibration table to minimize mechanical vibrations, a Faraday cage to shield against electrical noise, sound, and vibration, and a stand.

For the bilayer chamber, we use a circulating water jacket for temperature control in which the bilayer chamber is placed and a micro manipulator to mount the head stage of the patch clamp amplifier. An electronic filter unit is used to further filter the output from the patch clamp amplifier at 500 hertz. To visualize the current transitions, an oscilloscope is used for displaying the current signal, both the output from the patch clamp amplifier and from the electronic filter.

Last but not least, you will need a computer with a data acquisition system that digitizes the patch clamp signal. At 20 kilohertz, we use an in-house developed software for data recording and analysis. All of the equipment is turned on 20 minutes before beginning.

Next, we will prepare the chamber where the experiments are performed. The chamber is divided into two halves via septum, which is made of a thin Teflon sheet, about 100 microns thick and filled with an electrolyte solution. We form the bilayer across the hole in the septum, which provides for connection between the electrolyte solutions.

The hole should have a diameter of one to 1.5 millimeters using petroleum jelly as a sealant place. A glass window in the front half of the Teflon chamber, thread stainless steel screws through one metal brace and the four holes in the rear chamber half. Using petroleum jelly as a sealant place, a circular Teflon partition such that it will be sandwiched between the two halves of the assembled chamber.

Ensure that the hole in the partition is centered. Apply petroleum jelly on both sides of the partition to ensure that the electrolyte compartments are watertight and electrically isolated. Continue threading the screws through the front half of the chamber and the front metal brace.

Tighten the assembly to ensure that all components are held firmly in place, but not too tight in order to clean the chamber and remove the petroleum jelly. A pasture pipette is fire, polished and bent at a right angle.Fire. Polishing the pipette to a smooth tip helps avoid scratching the glass or Teflon.

Next, clean the chamber with a two to one mixture of chloroform and methanol or fch solution. Using the pipette, remove the excess petroleum jelly with the fch solution without smearing it all over the Teflon and glass. Use at least 10 volumes of fch.

Once this is done, magnetic stir bars are added to the chambers. Then clean the chamber with 95%ethanol. And finally with high purity water, either distilled water, UE water, or equivalent.

As before you use a fire polished pasture, your pipette to aspirate the liquid. Finally, repeat the cleaning with fch ethanol and water. Then water ethanol and fol.

Now we will make the glass electrode micro pipette, which will serve as the electrical connection to the rear compartment of the chamber using a P 97 flaming brown micro pipette Polar from Sutter instruments pulled the glass capillary to make two micro pipettes that each have a sharp and short taper. We use capillaries made of bo silicate glass with an external diameter of 0.8 millimeters and an internal diameter of 0.5 millimeters. A glass capillary is placed in the polar and fastened in preparation for pulling the filament in.

The polar should be made of platinum as we have not had success. Pulling the pipettes in electrode polars with tungsten filaments, the next few steps will be done in a micro forge with a platinum filament. Before using the micro forge, you should make sure that the filament is well coated with glass with.

You can either use a soft soda lime glass or a hard bo silicate glass. You will need to adjust the heat on your micro forge so that the glass flows smoothly and covers the filament uniformly with a thin coat. Now mount the micropipet in the micro forge and break the tip.

We do so by gently softening the glass coat on the filament at a relatively low heat. Then you gently move the micro pipette tip toward the glass coat on the filament so that the two melt together turn off the heat on the filament, which retracts and the pipette breaks. Now we are left with a sharp and even opening about 30 microns and diameter.

Again, using the micro forge, create a right angle bend in the micro pipette three to four millimeters from the tip, place the pipette a few hundred microns above the filament, and then turn on the heat so that the filament turns red. The filament will move due to the thermal expansion. Then gently lower the pipette onto the filament and the filament will bend.

You control the amount of bending by the heat of the filament. When the bend is about 80 degrees, you rapidly move the pipette up and forward. This requires some coordination.

You want to end up with a 90 degree bend using the same settings. Fire polish the micro pipette tip, keep the filament coated with glass. Now we siloized the pipette tip with a 10%solution of tri octal silene in benzene.

This makes the pipette surface hydrophobic so it interacts well with the membrane. Let the solution draw a few millimeters up into the pipette by capillary action. Evaporate the sane using the micro forge at low heat and you are done.

Now we will show you how to prepare the silver electrode. Take a five centimeter piece of silver wire about one millimeter in diameter. Roll it three to four times into a coil with an inner diameter of two to three millimeters.

Keep the rest as a tail which will bend such that it is at a right angle to the helix. Coat the tail and the adjacent five to 10 millimeters of the silver wire with molten silver chloride. Now prepare the bilayer forming solution.

In our case the lipid dial phosphatidylcholine in end decane. The lipid is stored in glass tubes at minus 40 degrees Celsius as 150 microliter al quads of lipid and chloroform. Take a glass tube from the freezer, let it come to room temperature and evaporate the chloroform under a jet of dry nitrogen.

In a chemical hood, add 50 microliters of end decane and vortex. To have good contact between the bilayer forming solution and the Teflon surrounding the hole over which we form the bilayer, we repaint the Teflon partition around the hole. First, clean a fire polished pasture pipette with fch.

Then dip the cleaned pipette into 2.5%phospholipid in end decane, allowing the lipid to rise about one millimeter up into the pipette. Deposit a drop of lipid as a film over the hole in the Teflon partition. Allow the lipid to dry for about 10 to 15 minutes and repeat.Hello.

So today we've shown you how to prepare lipid bilayers for single channel experiments. In the accompanying video, we will show you how to isolate small patches of membrane, how to record single channel activity, how to modify activity with small molecules, and how to analyze the results. Okay, that's it.

Thanks for watching. Good luck with your experiment.