This document provides instructions for analyzing AUC (analytical ultracentrifugation) data using the Ultrascan8.0 software. It describes how to edit raw data files from a Beckman centrifuge to produce a binary file readable by Ultrascan. It then explains the steps to analyze the data, including loading the data, selecting a model, checking diagnostics on individual curves, initializing parameters, floating select parameters during fitting, and performing the global fit.

1. Analysing AUC Data
Using Ultrascan8.0
Christiane Riedinger, October 2007
- Ultrascan manual:
http://www.ultrascan.uthscsa.edu/manual/edit_equil_abs.html
- Read also: my thesis! Appendix H… Karen Davies’ thesis a chapter about
AUC
organisation of raw data on Beckman Ultacentrifuge
- .ra1 = cell 1
- .ra2 = cell 2
- .ra3 = cell 3
- 0001 = wavelength 1 (270nm)
- 0002 = wavelength 2 (280nm)
- 0003 = wavelength 3 (330nm)
- 0001, 0004, 0007 = timepoint 1, 2, 3 of wavelength 1
- 0002, 0005, 0008 = timepoint 1, 2, 3 of wavelength 2
- 0003, 0006, 0009 = timepoint 1, 2, 3 of wavelength 3
1. edit data
you need to edit the raw data in order to produce a binary file containing the
entire dataset that can be read in by US.
- edit / equilibrium data/ absorbance data (brings up new window)
- click “select file”
- select data directory from Beckman centrifuge, the one containing the .ra1,
.ra2 and .ra3 files (launches new window)
- shows information about speeds, temperatures, wavelengths etc….
- enter run specification
- return to absorbance equilibrium data window
- now click edit data
- ignore the instructions, since they are wrong
- first enter centrepiece
- then click left and right of the meniscus
- again, click left and right of the meniscus, the program now zooms in onto the
meniscus
- then select the datasets you wish to include
- save
- repeat for each meniscus
- finally, the binary data file is saved which can be analysed (ending *.us.e)

2. 2. analyse data
- equilibrium / global fit
- things have to be done in this order, since some functions only become
activated when another one is performed!
- “load data”
- accept
- “select model”, e.g. 1 component, monomer-dimer… etc
- “scan diagnostics” performs a check on each curve, according to the following
criteria: Individual curves are excluded when the slope at the endpoint is too
low, indicating that there is little information content in the scan, when the
scan does not contain enough data points and when the curve does not exploit
the majority of the linear absorbance range (at least 0.6OD between 0.0 and
0.9 OD)
- manually check which curves have been excluded. You can force things to be
included if you have a good reason to do so
- “initialize parameters”: enter molecular weight and partial specific volume.
Load the file containing your buffer details for the density or enter the density
manually. Apply this to the scans you wish to include in your fit.
- “float parameters”: select which parameters you would like to float during the
fitting procedure. Obviously, you have to float the molecular weight! Make
sure that the boundaries are wide enough if your protein might be a dimer!!!
- “check scans for fit’: read through and see if there are any problems! This
activates the fitting control button
- “fitting control’: now you can perform your fit!(click ‘fit’)