Gramicidin-based Fluorescence Assay; for Determining Small Molecules Potential for Modifying Lipid Bilayer Properties

The overall goal of this procedure is to measure drug induced changes in bilayer properties as sensed by bilayer spanning proteins. This is accomplished by first making Fluor loaded, large unilateral sles. The second step of the procedure is to dope the vesicles with gramin.

The third step of the procedure is to add an external quencher and measure the rate of quenching of the internal fluoro.Four. The final step of the procedure is to quantify the change in Fluor quenching per gramin activity by fitting a stretched exponential function to the fluorescence time course. Ultimately, results can be obtained to evaluate whether amfi files can alter lipid bilayer properties.

Hello, I'm Ola Anderson. Hello, I'm Helson. Hi, I am Richie Kapo.

And I'm Leah Sanford from the laboratory of Dr.Olaf Anderson in the Department of Physiology and biophysics at Weill Cornell Medical College. Today we'll show you a procedure for measuring changes in lipid bio layer properties. We use this procedure in our laboratory to study the effects of drugs and small molecules on lipid bilar properties.

So let's get started On day one of this procedure, remove the lipid from the freezer and let it to equilibrate to room temperature. Once equilibrated, add 0.6 milliliters of 25 milligrams per milliliter, lipid and chloroform solution to a 25 milliliter round bottom flask. Then dry the solution under nitrogen gas while continuously rotating the flask until all of the chloroform has evaporated.

And a thin white film of lipid coats the entire lower half of the flask dry the lipid further in a desiccate under vacuum overnight. Prepare a solution of 100 millimolar sodium nitrate 25 millimolar, A NTS and 10 millimolar HEPs of pH seven. When adjusting the pH of this solution, avoid any chloride containing solutions.

As the thallium Quencher forms an insoluble complex with the chloride on day two, rehydrate the lipids in 1.67. One milliliters of this electrolyte solution to get a 10 millimolar lipid suspension. Once the correct concentration has been reached, cover with para film and vortex the suspension thoroughly protect the sample from light with foil and let it age at room temperature overnight.

On day three, sonicate the mixture for one minute in a low power sonicate. Then freeze thaw the sample with five minutes on dry ice, followed by five minutes in warm water, repeat the freeze thaw cycle four to five times. Extrude the lipid suspension using an Avanti mini extruder.

Set up the mini extruder with a 0.1 micron polycarbonate filter and filter supports extrude the suspension back and forth 21 times such that an almost translucent suspension ends up in the opposite syringe, resulting in a large ular vesicle or LUV suspension. Remove external A NTS by running the extruded suspension over a PD 10 duling column that has been equilibrated with sodium buffer. Add 1.5 milliliters of the LUV suspension plus one milliliter of sodium buffer to the column.

After the solution is fully incorporated into the column, elute the liposomes with three milliliters of sodium buffer and collect the eluate. The resulting A NTS filled LUV stock solution should contain about four to five millimolar lipid and appear translucent. Milky white.

Protect the solution from light with foil and store at 13 degrees celsius 24 hours prior to use thoroughly vortex. The A NTS filled LUV stock solution. As UUVs have a density greater than one gram per milliliter and or sediment.

Then dilute the A NTS filled LUV stock solution. One to 20 with sodium buffer incubate three quarters of the liposomes with 260 nanogram abidin in DMSO. Add the same volume of DMSO without gramin to the remaining one quarter.

To keep the solvent concentration constant in all samples, allow the gramin to equilibrate between the lipid vesicles inner and outer monolayers for 24 hours at 13 degrees Celsius. Here, an applied photophysics SX point 20 stopped flows spectro fluorimeter with temperature control is used to measure the rate of fluorescence quenching. To begin turn the instruments on an hour ahead of time to allow for instrument warmup.

The slit with rapporteurs for excitation should be one slash one and a 4 55 nanometer high pass filter should be used for recording emission using the pro data SX software. Set up the recording conditions first, set the monochromator excitation wavelengths to 3 52 nanometers. Use the pressure hold setting.

Set time to one second and points to 5, 000. Adjust the number of repeats to nine for buffer runs and to 13 for quencher runs. Monitor the temperature in the Essex software check drive profile.

The drive volume should be set to 120 microliters, which mixes 60 microliters from each syringe. This gives a dead time of approximately 1.2 milliseconds. Adjust the high voltage or gain on the fluorescence detector to about 420 volts.

The goal is to obtain a fluorescence reading of about eight equilibrate the previously prepared A NTS loaded UUVs for 10 minutes at 25 degrees Celsius in the dark. Once equilibrated vortex the sample, then load it into the left syringe. In the right syringe load, either sodium buffer or thallium.Quencher.

Remove any air bubbles by pushing the syringes back and forth. Both syringes should be loaded equally. Before closing the valves for the very first sample, record fluorescence with sodium buffer.

Repeat four times and adjust the gain as necessary. Then repeat an additional five times for the remaining samples. Record nine repeats with sodium buffer.

Replace the sodium buffer with the thallium quencher and record 13 repeats. Rinse with water and continue with the next sample controls. Include samples with no gramin and with solvent, with no gramin and the maximum compound concentration.

And with both gramin and solvent following the fluorescence assay, read the data into the MATLAB software for analysis. For each sample read in all the buffer and quencher repeats, the first four repeats will be contaminated by what was previously in the tubing. In this case, the signal comes from the sodium buffer sample mixing with water from the previous wash.

Repeats five through nine represents signal from the sample alone. Therefore, we always exclude the first four repeats in each case. To avoid any mixing artifacts, manually go through the traces and remove any bad repeats, such as the red trace containing spikes or deviations due to mixing artifacts and or bubbles.

Then normalize the quencher repeats to the average starting value of the buffer repeats for each sample for each experimental condition, combine averages of all the samples into one graph of visualization samples with no gramine should all be similar and show almost no fluorescence quenching. There is a slow reduction in the fluorescent signal due to slowly because of thallium ion into the vesicles for samples with gram sidin. If the fluorescent time course is visibly altered by the compound, then the compound alters lipid bilayer properties at the concentration tested next fit a stretched exponential to the first two to 100 milliseconds of the normalized fluorescence quenching curves.

For the individual repeats and calculate the rate at two milliseconds. Then calculate the averages and standard deviations for a given sample from the individual Repeats the effect of capsaicin denoted here as cap was observed on the time course of fluorescent quenching in the absence or presence of gramin denoted as ga, the normalized fluorescent signal is shown over one second here. Gray dots represent results from all repeats while the red lines are the averages.

The first 100 milliseconds of the normalized fluorescent signal is shown. Gray dots are results from a single repeat for each condition. Red lines are stretched exponential fits to those repeats.

The stifled blue line denotes the two millisecond mark. The time at which the rate of quenching is determined. The relative change in quench rate is determined by normalizing the data to the control sample with gramin and no modifier.

When capsaicin absorbs to the lipid bilayer, it alters the bilayer properties. The change towards the softer bilayer will be reflected as a shift in the gram side in mono dimer equilibrium in favor of the conducting dimer. We've just shown you a procedure to measure the bilayer perturbing effects of a small molecule.

When doing this procedure, it's important to remember to use the proper controls for each experiment using the same batch of vesicles. So that's it. Thanks for watching and good luck with your experiments.