“Patience is a virtue” Chinese proverb Welcome to Analytical Ultracentrifugation Where

Outline of topics Care and use of cell components and rotors Care and use of the instrument Using the Beckman data acquisition software

Cell Components ● Windows – Sapphire ● Must be used with Rayleigh interference optics ● May be used with absorbance optics at wavelengths above 240 nm ● Each window weighs approximately 5.8 grams – Quartz (softer and easier to break than sapphire) ● Used with absorbance optics at all wavelengths ● Can not be used with Rayleigh interference optics ● Each window weighs approximately 3.8 grams ● Must be used with fluorescence optics

Cell Components ● Centerpieces: always check Beckman's or other manufacturer's Compatibility Table before choosing centerpieces – Epon-charcoal, 2-sector ● Compatible with a wide range of solvents ● Sedimentation equilibrium & velocity experiments ● Speeds up to 42,000 rpm – Aluminum, 2-sector ● Can not be used with high salt buffers or buffers containing strong acids/bases ● Speeds up to 60,000 rpm ● Must use gaskets on both sides of the centerpiece

Cell Components ● Centerpieces – Titanium, 2-sector ● Compatible with a wide range of solvents ● Speeds up to 60,000 rpm ● Requires two gaskets ● Requires titanium counterbalance – SedVeloc60K, 2-sector (CAMIS) ● Speeds up to 60,000 ● No gaskets required

Cell Components ● Centerpieces – Band forming ● Used when small amount of sample available ● Uses buffer of significantly higher density i.e. D 2 0 – 3 mm Centerpieces ● Used for high concentrations of protein ● Used for small sample volumes ● Spacers are required

Cell Components ● Centerpieces – Epon-charcoal, 6 sector ● Sedimentation equilibrium ● Maximum speed is 48,000 rpm ● Shorter columns ● At 42,000 rpm, the g force exerted at 6.0 cm (inner chamber) is while the g force exerted at 7.0 cm (outer chamber) is – Titanium, 6-sector (Nanolytics) ● Sedimentation equilibrium ● Maximum speed 60,000 rpm ● Requires special gaskets and titanium counterbalance

Other Cell Components ● Window Assemblies (2 per cell) – Window holder – Window gasket (Vinylite) – Window liner (Bakelite) ● Cell Housing ● Housing plugs & housing gaskets (2 per cell)

A Special Cell – The Counterbalance ● Anodized red ● Positioned in cell hole 4 of An-60 Ti rotor or hole 8 of An-50 Ti rotor with the more widely spaced reference holes to the outside of the rotor ● A set screw on the top is loosened or tightened as necessary to remove or secure the counterbalance in the hole with alignment of scribe marks on rotor and counterbalance ● The radial positions of the counterbalance are known and reported to the software for radial calibration in absorbance mode and is used to visually set the inner and outer radial calibration positions using the interference optics

Counterbalance ● Weights are added to the counterbalance to balance the opposing cell and must not protrude from the top or bottom ● Without added weights, the weight of a counterbalance is ca. 32 grams

Cell Assembly (2 or 6 sector) ● Notice the keyway on the window holder ● Place gasket into the holder Note: replace gaskets and liners as needed ● Place liner in the holder with open side opposite the keyway ● Place the window into the holder aligning the arrow on the window with the keyway ● You need two assemblies per cell Keep a supply of purified nitrogen gas nearby to blow of dust during the cell assembly process

Cell Assembly (2 sector) ● With the open side of the window facing up, place the first window assembly into the cell housing using the key for alignment and push it to the bottom of the cell ● Place the centepiece into the cell housing with the part number facing upright using the key for alignment ● Place the second window assembly into the holder with the open face of the window facing downwards

Cell Assembly (2 sector) ● Very lightly lubricate the screw ring washer and the screw ring with SpinKote on a periodic basis ● Insert the washer ● Insert the screw ring and hand tighten ● Torque the entire assembly to 120 inch-pounds

Loading 2-Sector Centerpieces ● The easiest way is to piggyback a rounded gel loading tip onto a 1000 µl tip ● You can also use a Hamilton syringe and polyethylene tubing – avoid needles! ● Place the cells with the screw ring facing forward and the fill hole on top ● The reference (solvent) is placed in the left fill hole and the sample in the right ● For runs done in absorbance mode, load 10 – 20 µl more reference than sample

Loading 2-Sector Centerpieces ● Place small polyethylene plug gasket over each hole ● Screw small brass plugs into holes and tighten by hand ● Do not overtighten plugs or the housing will become distorted ● Weigh cells placed in opposing holes in the rotor to insure the weights are within 0.5 grams of each other

Assembling and loading 6 sector centerpieces ● The cell housing requires two screw rings and gaskets unlike the double sector cells ● Note the centerpiece has one end with a narrow base: orient the cell housing with the part number upside down and insert the centerpiece into the housing ● Place a window assembly onto the end with the centerpiece and insert the screw ring gasket and screw ring ● Torque to 60 inch-pounds

Assembling and Loading 6 Sector Centerpieces ● Orient the cell with the cell housing part number right side up and the fill holes facing left ● The reference buffer is placed into the front sectors (ca. 120 µl) remembering that the left sector will be toward the inside (less force) ● The samples (110 µl each) are placed in the back sectors ● The top window assembly is placed along with the top screw ring gasket and screw ring; torque both sides to 120 inch-pounds

Rayleigh Interference Optics ● Always use a matched buffer i.e. dialysis buffer as the reference buffer because of gradients that form ● Match the menisci of the sample and reference sectors We're almost there!

Rotors ● An-50 Ti (8 holes) and An-60 Ti (4 holes) are the two standard rotors used in AUC ● An overspeed ring or disk is located on the bottom and must be aligned correctly ● Two small magnets are located in the ring and must be present and intact: read by the Hall effect sensor, they convey information about the position of hole #1 ● An alignment tool is available to properly align the correct replacement ring

Placing Cells in the Rotor ● With the screw ring up (on two sector cells) and the fill holes facing the inside of the rotor, push the cells into the appropriate rotor hole ● For six sector cells, the cell housing part number should be right side up and the fill holes facing the inside of the rotor ● Using the cell alignment tool, align the scribe mark on the bottom of the two sector cell with the scribe mark on the bottom inside of the rotor: use a mirror or place the rotor carefully on its side to aid in alignment ● The six sector cell does not have a scribe mark; however, the keyway is visible so align the center of the keyway with the scribe mark on the bottom inside of the rotor ● Proper alignment is important so that the baselines and plateau regions of the data are horizontal

Cleaning Cell Components ● Disassemble the cells using the torque wrench ● Rinse the windows and centerpieces with water ● Place the windows and centerpieces in a sonicator containing a mild detergent such as liquinox ● Sonicate for minutes ● Using powderless gloves, remove the windows and gently wipe, rinse with ethanol, and rinse with RO/Milli-Q water. Stand on lens paper. Rinse the centerpiece in a similar manner without wiping ● Gently wipe the windows with good quality lens paper and allow the centerpiece to air dry. Use purified nitrogen gas to accelerate the drying process and to blow off dust from the windows and centerpieces while assembling ● Special cleaning for handling RNA is required

Beckman XL-I

Use and care of the XL-x ● Themoelectric heating and cooling with a temperature range of 0 – 40°C ● Speeds of 3 to 60 krpm ● Optical systems – Absorbance: 190 – 800 nm – Rayleigh Interference: 675 nm – Fluorescence: 488 nm

Rotor temperature Overspeed system Magnetic Position Sensor 3 or 5 pin Mechanically changes radial position in absorbance mode For interference optics

Care of the interior and exterior ● Use a mild soap to wipe the inside of the bucket periodically. Oils will build up especially around the monochromator mount. Be careful to avoid the slit assembly, the condenser lens, and the radiometer when cleaning. A mild soap can also be used to clean the exterior. ● Clean around the lid and top O-ring. Apply vacuum grease (just enough to make it shiny) as necessary. ● You may want to apply a thin layer of SpinKote to the monochromator mount if it becomes difficult to tighten the monochromator onto the mount. ● There are no user servicable items in the bucket. Check with your Beckman service engineer before attempting to remove the safety plate and examining/cleaning the slit assembly or the underlying photomultiplier tube.

The Monochromator ● The monochromator selects the wavelength of light to pass through the sample ● In the XL-I, laser for the Rayleigh interference optics is attached to the monochromator ● A lever on the side of the monochromator can be set to work at wavelengths below or above 400 nm

The Monochromator ● Monochromators for the XL-A have 3 pins while monochromators with attached laser for interference optics have 5 pins ● There are two guide pins ● The wavelength selector pin extends from the mounting receptacle and must be retracted before mounting the monochromator

Xenon Flash Lamp ● The xenon flash lamp should be cleaned every 4 – 6 runs ● To remove the flash lamp, first remove the console cover and the front cover of the instrument ● Turn off the power to the instrument and unplug the power cord from the power supply ● Mark the front of the lamp assembly (use a piece of tape); using an appropriate sized Allen wrench, loosen the hex screw holding the lamp assembly Allen wrench

Xenon Flash Lamp ● Carefully remove the lamp assembly ● Remove the hex screw holding the cap in position ● Mark the position of the cap relative to the rest of the assembly and unscrew and remove the cap being careful not to lose the O-ring inside ● You can use a pencil eraser or some toothpaste on a KimWipe or Q-Tip to remove the build-up on the top surface of the flash lamp ● Repeat if necessary

Xenon Flash Lamp ● Then wipe the surface with ethanol on a KimWipe or lens paper to help remove all the toothpaste or erasings. ● Wipe with a little Milli Q water because some alcohols leave a little residue ● Polish with lens paper and replace the cap (while making sure the O- rings stay in position) ● Align the cap with the assembly; replacd the hex screw and tighten

Xenon Flash Lamp ● Place the lamp assembly in the instrument aligning the tape on the assembly with the front of the instrument ● Tighten the hex screw, replace the front cover, and the console cover ● The lamp assembly can be rotated in order to maximize the intensity and minimize intensity variation across the length of the cell ● Hence, the tape keeps the same alignment after cleaning the lamp

Data Acquisition Software

Notice this is performed at 3000 rpm so there is no waiting for vacuum or temperature. We are acquiring intensity data and doing a radial calibration before the scan. The instrument is set to stop when the scan is completed.

230 nm peak is off by several nm as is the 527 nm peak

Click on Select Peaks

Move arrows to misaligned 230 and 527 nm peaks

Click on Save Peaks and Press Enter

Re-run the original scan and see that the 230 and 527 nm peaks are in the proper position.

Using a windowless cell, you can do a velocity scan (3000 rpm) collecting intensity data. This will provide information about variations across the cell. You can do these checks at different wavelengths.

We are going to set up a sedimentation velocity experiment in absorbance mode of protein DoD, running at 60,000 rpm, 20°C, with a radial step size of cm, collecting 100 scans as rapidly as possible. Follow the shaded areas..

You can also open a pre-existing file.

Be careful! The conditions of the previous run are shown in the XL Settings.

You may wish to change Rmax to 7.2 cm. No information is found beyond 7.2 cm.

Click on stop XL after last scan and overlay three scans (you may prefer more or less)

Using a radial step size of cm is recommended unless you want to collect scans faster.

After unchecking, you can then make changes to the other Comments.

Save scan for future runs!

Start Method Scan

Once you have started a method scan, double-check the conditions such as temperature, selected speed, the number of scans, etc. You must stop the method scan before making any changes to the method and then re-start. Once the vacuum is 50 microns or below and the temperature has equilibrated to the set temperature, then press the start button on the centrifuge console to begin the run.

Trouble shooting If there is a rise in temperature and vacuum while the centrifuge is accelerating, a leak has probably occurred or a cell has broken. Stop and inspect. If the instrument fails delay calibration, check that the correct rotor has been selected in the XL Settings. If you have changed rotors or counterbalances since the previous run, a radial calibration must be performed prior to the current run. If your data is upside down, the cells were either loaded incorrectly or they were placed in the rotor backwards.

From Interference menu, choose Radial Calibration. The rotor has to be balanced and spinning at 3000 rpm.

From this menu, go to Laser Setup

Adjust Laser delay, Laser duration, brightness and contrast. You should be able to see the fringes of the counterbalance clearly.

Laser delay: sets the angle and can be done automatically or manually Laser duration: how long the laser fires; generally set at 0.6 Brightness: generally set at 1 Contrast: generally set 127 You want the clearest fringes possible – clear, alternating black and white lines.

Move the cursor to a point on the left fringe area (inside at 5.85 cm) where the white and black fringes end. Click there and then click on Inside and Set Radius in the Radial Calibration box. Do the same for the outside (7.15 cm) fringe area.

There are several differences between velocity runs performed with interference optics as opposed to absorbance optics. 1.A setup (3000 rpm) must be performed on each cell prior to the run to insure clarity of the fringes. 2.Interference scans occur much more rapidly because there is no mechanical movement of the slit assembly. Approximately 10 sec is required for each scan. Therefore, you must determine how many scans are necessary and if time between scans is required. 3. Optical problems may occur at all temperatures but especially when running at low or high temperatures because oil vapors condense more readily on the camera filter which is visible on the bottom of the can. 4.You may want to check the laser delay after the rotor has reached speed. There may be a need to correct for rotor stretch at high speeds.

Prior to multi-wavelength equilibrium experiments in absorbance mode, wavelength scans are performed to use later for determining the extinction coefficients at the measured wavelengths.

We need a wavelength stepsize of 1 and 1 replicate.

We want to run the scan 3 times at 3000 rpm for each radial position. For 2-channel centerpieces, that would be 7.0 cm, but 6-channel centerpieces, scans need to be taken at 6.0, 6.5, and 7.0 cm assuming all chambers are used.

Typical setup for equilibrium scans in absorbance mode. We will be using 6-channel centepieces in a An60Ti rotor. If you use 2- channel centerpieces and use 120 µl of samle, set the Rmin to 6.8 cm.

Set the radial set size to and the number of replicates to 20. Insert the data directory name. IMPORTANT: Even though we are using a 6 channel centerpiece, UltraScan requires us to select a 2-channel centerpiece.

Enter the speeds, delay conditions, temperature, and number of scans in the Method. Delay conditions are determined in the UltraScan program as are the speeds. Above is a typical setup. Note that duplicate speeds are used.

Finally, select the appropriate wavelengths for each cell. In this case, we are reading cell 1 at 280 nm, cell 2 at 230 nm, and cell 3 at 208 nm. Start Method Scan

A “Glowing” word about FDS – the fluorescence detection system. This evolving technology has recently become available and we are currently in the process of evaluating it.

Several technical points: Quartz lenses must be used High temperatures (above 30°C) “cloud” the optics Samples should be degassed before running There is no blank, so both channels can be used for samples The 10-channel calibration cell is necessary

Control Box