Hi, I'm Andrea Balbo. I work in Peter Shucks group at the National Institutes of Health. In this video, we're going to demonstrate to you our procedures for the assembly, loading and rotor alignment of an analytical ultracentrifuge sample cell.
Let's get started.Collected. Here are the components of an analytical ultracentrifuge sample cell Stacked inside the cell housing is a 12 millimeter double sector charcoal field, epon centerpiece that will be sandwiched between two window assemblies. Each window assembly consists of a window gasket, a window liner, and a window held inside the window holder.
These pieces will then be slid down inside the cell housing by aligning their keyways with the key that's found inside the housing barrel, a housing gasket, and then a screw ring closed the sample cell with a torque pressure of between 120 and 140 inch pounds. After loading your sample in reference two housing plug gaskets and a housing plug seal the loading holes of the cell to avoid scanning artifacts, leaks, and breakage during experiments, it is vital that all components be clean, dry, dust, free, and in the best possible condition, examine often, and replace when necessary. You will also need the following, an analytical ultracentrifuge, titanium four hole rotor, a laboratory balance, a torque wrench with a readable dial and a torque stand, A counterbalance and counterbalance weights, A flathead screwdriver, a pair of forceps, a 200 microliter pipetter fitted with a long tapered pipette tip, alignment tools and spin coat lubricant.
We begin sample cell construction by putting together two window assemblies. First place a window gasket at the bottom of the window holder. Next position the window liner, or sometimes it's called a window cushion inside the window holder so that the opening of the liner is opposite the keyway of the holder at a slight angle.
Then place the window in the holder, aligning the scribe mark on the window with the keyway of the window holder. Press down gently at the very edges on both sides of the window. To ensure proper sealing and accurate twerking of the sample cell, we lightly coat the housing gasket in the screw ring.
With spin coat lubricant, spread a very small amount of spin coat between your thumb and forefinger. Coat the screw ring threads with a thin, invisible film of spin coat. Likewise coat the housing gasket.
Wipe off any visible lubricant. To begin sample cell assembly. Slide one window assembly with the window facing up towards you into the cell housing by aligning the keyway with the housing key.
Align the centerpiece keyway with the housing key and let it gently fall on top of the window assembly inside the housing. Never use any sort of tool to push the centerpiece into the cell housing. This could cause permanent damage to the centerpiece resulting in leak storing experiments.
Turn the second window assembly so that the window is facing towards the centerpiece and away from you. Align the keyway with the housing key and slide it down on top of the centerpiece. Place the housing gasket on top.
Then a screw ring so that the word out is visible. Using your fingers. Then an alignment tool, hand tighten the screw ring.
Sometimes the centerpiece doesn't slide easily into the housing barrel. You can first align it, then place the second window assembly on top of it. By applying gentle downward pressure on the window assembly.
You can slide both into the cell housing at the same time. In this way, we avoid pressing directly on the centerpiece. Now is a good time to look the cell for dust lint and fingerprints.
You will also notice Newton's rings that indicate there is still air between the centerpiece and the windows. These will disappear after torquing with the screw ring up and the word out visible. Place the sample cell all the way down inside the cell.
Torque collette of the torque stand. Hold the cell in place by applying constant pressure on the torque stand handle. In one continuous motion, torque the screw ring to between 120 and 140 inch pounds.
Release the torque stand handle and carefully remove the sample cell. We routinely load a volume of 400 microliters into each sector of the 12 millimeter centerpiece. Using a 200 microliter pipette position the sample cell with the loading holes on top and the word out on the screw ring visible.
Slide the pipette tip halfway down into the left centerpiece sector through the loading holes and slowly dispense 200 microliters of reference solution. Repeat once to total 400 microliters using the same pipette tip and the same technique carefully and slowly. Fill the right centerpiece sector with 400 microliters of sample solution.
It is imperative to match the volumes of reference and sample solutions as closely as possible. Fitting the pipette tip firmly onto the pipette, slowly loading and dispensing solutions, avoiding loading bubbles, and using the same pipette tip for both solutions. All aid in keeping the volumes equal to seal the cell.
Place two red housing plug gaskets snugly into each loading hole, making sure they are positioned carefully between the centerpiece and the aluminum housing. Cover each loading hole with a housing plug and hand tighten with a small flat head screwdriver. Before loading the rotor, make sure the counterbalance and the cell that will be opposite the counterbalance are within 0.5 grams of each other in weight.
Start out with a balance at zero. First, weigh a sample cell that will be in position opposite the counterbalance. Tear the balance to subtract the sample cell weight.
Then weigh the counterbalance. Here we find the counterbalance to be 4.193 grams lighter than the sample cell. Select a four gram counterbalance weight and screw it down into the attachment hole of the counterbalance, making sure it does not protrude from the top.
Reigh the counterbalance to make sure the final weight is within 0.5 grams. Using a four hole rotor, place the counterbalance into position four so that the white arrow is visible and pointing in the direction of centrifugal force. Using an alignment tool, make sure that it's all the way down inside the rotor.
Position the balance sample cell with the housing plugs facing the center of the rotor and the word out on the screw ring visible. Again, using an alignment tool, make sure it's all the way down inside the rotor. Looking from underneath the rotor.
Align the inside. Scribe, mark the mark closest to the center of the rotor with the scribe mark on the counterbalance Using an alignment tool. In the same way, align the sample cell at position two.
After all the cells are aligned, the rotor is ready for placement in the centrifuge chamber.
