This method can help study directions of proteins with lipid membranes. In our case, use it to analyze the calcium dependent binding of annexins to negatively charged phospholipids. The main advantages of this technique are that it's label free, sensitive, quantitative, and that interactions can be observed in real time.
Thus allowing direct analysis of real results. This technique can also be applied to other systems, such as the interaction of more complex micromolecule assemblies or even cells. Use clear five millimolar lipid solutions as described in the text protocol.
Combine the dissolved lipids in the desired molar ratio in 10 milliliter glass tubes. Evaporate the organic solvents using a dry stream of nitrogen. Leave the lipid mixture on a high vacuum lyophilization system for three hours to remove any residual traces of the solvents.
Now, resuspend the lipid film in one milliliter of citrate buffer. Incubate the lipid suspension at 60 degrees Celsius for 30 minutes in a water bath, vortexing it vigorously every five minutes. This temperature is around 10 degrees Celsius above the phase transition temperature of the highest melting lipid in the mixture.
Meanwhile, preheat the extruder equipped with a 50 nanometer diameter pore sized polycarbonate membrane above the transition temperature for 30 minutes. Load the multilamellar vesicle suspension into the preheated extruder. Gently pass the mixture 31 times through the polycarbonate membrane to form small unilamellar vesicles or SUVs.
Keep the temperature above the transition temperature. Transfer the SUV suspension to a two milliliter plastic reaction vessel and add the citrate buffer to bring the final volume to two milliliters. Incubate four sensors inserted in a polytetrafluoroethylene holder in a 2%SDS solution for at least 30 minutes.
Then wash them extensively with ultra pure water to completely remove the SDS and try them using a stream of dry argon or nitrogen. Use a plasma cleaning system to completely remove any contaminants. To do so, insert the dry sensors in the plasma cleaning chamber, evacuate the chamber, and flush it three times with oxygen.
Then, turn the plasma cleaner on. Use vacuum, high radio frequency power, and 10 minutes of process time. Following the cleaning run, turn off the machine and take out the sensors.
Carefully dock the plasma cleaned sensors into the four flow chambers using tweezers. Avoid any pressure on or torsion of the chambers and tubes that may cause leaking. Flush the system with citrate buffer in the open flow mode for 10 minutes.
Launch the program. Start recording any changes in the frequency and dissipation of the first fundamental tone and overtones using the software until the frequency and dissipation baselines are stable. When the baselines are stable, apply the SUV suspension in citrate buffer.
Using a reaction vessel, remove 1.5 milliliters of the dead volume, then close the system in the loop flow mode. Record the frequency dissipation shift for another 10 minutes. When the SLB is stable, equilibrate the system with a running buffer at the required calcium concentrations in an open flow mode for 40 minutes.
Add the protein to the running buffer containing calcium. Perform the application of the protein in a loop flow mode until an equilibrium steady state is reached. Now, dissociate the bound protein by chelating calcium ions with five millimolar EGTA in the running buffer in open flow mode.
Regenerate the microbalance system with 50 milliliters of double distilled water in a continuous open flow mode. Remove the tubes from the water container and let the system run dry. Carefully remove the crystal sensor and clean it with a 2%SDS solution using the polytetrafluoroethylene holder.
Dry the visible parts of the flow module interior where the sensor was placed. Shown here, is the recording of the frequency curve and dissipation shifts. The prominent drop in frequency upon the addition of the liposomes, indicates their absorption because the buffer filled vesicles are not rigid but viscoelastic, the dissipation increases.
Subsequently, the coalescing vesicles rupture. The concomitant release of the buffer inside the vesicles decreases the adsorbed mass until a stable plateau is reached. The binding of Annexin A2 to the lipids adds mass as seen by the clear frequency shift, but does not interfere with the bilayer structure as indicated by the only small change in dissipation.
When calcium ion is removed by the chelating agent, EGTA, Annexin A2 dissociates from the lipid film. The frequency and the dissipation recordings shift to the levels seen with the bilayer only indicating the Annexin A2 binding is totally dependent on the calcium ion and that the lipid film remains intact. A representative negative control experiment is shown here.
When phosphatidylserine is absent, no changes in frequency or dissipation are apparent after addition of Annexin A2 in the presence of the calcium ion. While attempting this procedure, it's important to ensure proper bilayer formation. Use the liposomes immediately, otherwise the small vesicles will fuse into bigger ones with less surface tension that can lead to an unstable baseline.
Following this procedure, a quantative description of a membrane protein interaction can be performed in order to answer additional questions like cooperative versus non-cooperative binding.
