The overall goal of the following experiment is to determine the size of the oligo formed by a small hydrophobic polypeptide in C 14 sulfalane detergent and the stability of this olier when pH is changed. This is achieved first by determination of which deuterium oxide concentration matches the density of detergent my cells, and thus neutralizes their buoyancy. As a second step, a set of sedimentation equilibrium experiments for the small hydrophobic protein samples solubilized.
In C 14 sulfalane detergent is run with the determined deter oxide concentration. Next, the first and second steps are repeated at a different pH in order to compare the sedimentation profile of the protein at these various pH values. The results will show both the oligomeric size of the small hydrophobic protein in this detergent and the effect of pH on the stability of this oligo in C 14 sulf beane detergent.
Visual demonstration of this method is critical because several, there are several steps involved like assembly and loading the cells density matching and reaching equilibrium conditions, and this can easily overwhelm and confuse first time users. Any small mistake in these steps can potentially waste several days worth of experimental time. The procedure will be demonstrated by suria, a PhD student in my lab.
First, prepare 10 x buffer and 50 x detergent. Stock solutions and filter sterilize each through a 0.2 micrometer syringe filter into a 1.5 milliliter einor from the 10 x buffer and 50 x detergent stock solutions. Prepare 200 microliters of three separate sample solutions containing 10%30%and 50%deuterium oxide by volume.
To assemble a six channel analytical ultra ification cell without external fill, place the window gasket into the window holder slightly bend the window liner and place it into the window holder such that the gap is formed opposite to the window holder. Keyway then place a sapphire window inside the window liner, aligning the mark with the window holder. Keyway place to sell housing with a part number upside down with the keyways in line with the housing key.
Slide a six sector centerpiece with beveled side down into the cell housing followed by one window assembly with a window facing down Next lightly coat the screw ring threads and screw ring washer with spin coat lubricant. Place a screw ring washer on top of the window assembly. Install the screw ring into the window housing with the word out facing outward hand tighten the screw ring by using the cell aligning tool.
Then tighten the screw ring to only 60 inch pounds using the torque wrench. Following this place a cell with the part number upright and position at 12 noon. Load 120 microliters of reference solution into the left rows and 110 microliters of sample solution into the right rows.
Using a 200 microliter pipette ensuring that each sample and reference is correctly paired carefully. Slide one window assembly with the window facing down into the cell housing, taking care not to disturb the cell excessively as this could spill their contents. Repeat the previous steps, tightening the second screw ring to 120 inch pounds.
Then invert the cell and re-tighten the first screw ring to 120 inch pounds. Load the cells into the rotor. Install the rotor into the centrifuge, and install the monochromatic according to the manufacturer's instructions.
After sufficient vacuum has been reached, run the centrifuge at 3000 RPM. Preview the interference pattern in the user interface software and adjust the laser parameters to obtain the highest contrast. Next, create a new setup file, specifying equilibrium and interference measurement.
Set up a sedimentation equilibrium method to run at 45, 000 revolutions per minute or the highest speed anticipated for protein samples with a run temperature at 20 degrees Celsius After collecting one, scan every 15 minutes for at least 12 hours. Monitor equilibrium progress by opening the data files in hetero analysis and selecting match function. At this point, assemble a six channel analytical ultracentrifugation cell with quartz windows in the same manner as before.
Using gel loading tips, load the previously prepared highest concentration small hydrophobic protein sample in the channel nearest to the rotor center, and lowest concentration sample furthest from the rotor center. To set up the absorbance measurement, create a new setup file specifying equilibrium and absorbance measurement. Specify 280 nanometers as the detector wavelength.
Following this, perform a radial calibration at 3000 RPM by checking radial calibration before first scan and scan options. Specifying data collection at low resolution and executing a single scan. After the scan is complete, uncheck the option.
Set up a sedimentation equilibrium method to run at the first speed previously calculated at 20 degrees Celsius and collect one scan every 30 minutes in each cell's detail. Specify data collection at low resolution after at least 18 hours. Monitor equilibrium progress by opening the data files in hetero analysis and selecting match function once equilibrium has been reached.
Collect a single scan at high resolution after the scan is completed. Repeat the previous steps for the next speed when the time required to reach equilibrium for each speed is known. Set up the sedimentation equilibrium method Scan to include all the speeds previously calculated and to collect one scan after the equilibration time for each speed.
In this case, specify high resolution data collection in each cell's detail the radial distribution of C 14 sultan detergent my cells, and 50 molar sodium phosphate buffer. pH 7.3 forms a very shallow exponential that could be fitted to a linear model. The slope of this profile depends on the deuterium oxide concentration and the concentration that produced zero slope was found to be 32.3%The same experiment was repeated for pH 5.5 and pH three to obtain matching deuterium oxide concentrations of 30.3%and 41%respectively.
At pH 7.3, the small hydrophobic protein and detergent was found to form pentamers with apparent logarithm of the affinity constant of 21.05. The association constant did not change at pH 5.5, but it was significantly reduced at pH three. This is consistent with previous reports showing a decrease of conductance at lower pH with a PK of approximately 4.5.
Once master at equilibrium cell assembly, loading and experiment set up can be done in one hour if it is performed properly. After watching this video, you should be able to have a good understanding of how to prepare a sedimentation equilibrium experiment so that you can determine the oli matic size of your membrane protein and also to compare stability and their different experimental conditions.
