SEDFIT-SEDPHAT workshops at AUC2009 in Uppsala,
September 14/15 2009
There will be two SEDFIT-SEDPHAT workshops in parallel. The workshops are a mixture of theory lectures and hands-on data analysis
exercises on your own laptop. They will take place from 9:00h on Monday,
September 14 to 18:00 h on Tuesday, September 15. The instructors
are Chad Brautigam, Patrick Brown, Rodolfo Ghirlando, and Peter Schuck. For more detailed information about the
workshops and registration, please contact
schuckp@mail.nih.gov.
SEDFIT-SEDPHAT - Analysis of Non-Interacting
Systems by SE, SV, and DLS
SEDFIT-SEDPHAT - Global Analysis of Interacting
Systems by SV, SE, ITC, and SPR
There are still spaces available. For
registration, please follow the registration instructions at the
conference website
AUC2009.org, and please independently send me an email
to schuckp@mail.nih.gov
so that we can follow the registration and be sure to send
you material in preparation for the workshop.
SEDFIT-SEDPHAT - Analysis of Non-Interacting
Systems by SE, SV, and DLS
This workshop is focused on tools for the characterization of
non-interacting systems, including determining mass, size, size-distributions,
hydrodynamic shapes, and trace components. The experimental setup of analytical
ultracentrifugation experiments will be discussed with regard to the inseparable
correspondence between experimental design and reliable data analysis. The
principles of dynamic light scattering and its data analysis in SEDFIT are
presented. A significant segment of the workshop is dedicated to the modeling
with distributions. The application of the different forms, the limits of
resolution, and the confidence in detailed interpretations will be discussed.
New developments are presented, including the Bayesian distribution analysis,
partial boundary modeling, and buffer mismatch modeling.
The workshop is a series of alternating lectures and guided
computer exercises by the participants on their own laptop. This workshop is
suitable for beginners as well as experienced SEDFIT/SEDPHAT users.
Preliminary
Agenda:
·
Introduction to AUC and overview of
parameters for designing and running an experiment
o
Considerations for the choice of optical
system, rotor speeds for SV or SE, buffers, concentrations, partial-specific
volumes
·
Introduction to DLS
·
Theory and practice of sedimentation
velocity for non-interacting systems
o
Introduction to noise elimination, Lamm
equation ls-g*(s), c(s,M), c(s,*), c(s), c(M)
o
Practical exercises for basic SEDFIT:
loading data, using the different models, quality of fit, integrating
distributions, buffer corrections, documenting results
·
Tools for analyzing DLS data in SEDFIT
o
Importing data, Stokes-radius distributions,
discrete species
·
Optimization and Statistical Error Analysis
o
Introduction to minimization routines, error
estimates by covariance matrix, Monte-Carlo simulations, F-statistics
projections, error analysis for distributions, regularization, Bayesian analysis
o
Practical exercises: determining a
confidence interval of a single parameter, distributions with
Bayesian prior
·
Introduction to SEDPHAT
o
Principles of organization
o
Practical exercise transferring data between
SEDFIT and SEDPHAT, configurations, hybrid discrete/continuous model, links of
local parameters
o
Partial boundary modeling
·
Tools for the characterization of trace
components
o
Application of c(s), confidence, statistical
error analysis, standardized integration, serial analysis, Bayesian adaptation of regularization
o
Dynamic density gradients
·
On particle-solvent interactions in
analytical ultracentrifugation
o
effects of hydration and solvation in the interpretation of buoyant molar mass
and hydrodynamic friction
o
membrane proteins, nucleic acids,
denaturants, osmolytes
·
Special features of SEDFIT and SEDPHAT
o
Buffer mismatch modeling
o
Overview of models and utility functions
·
Basic hydrodynamic modeling with HYDROPRO
·
Oops – Don’t/Do Panic? (things that can go
wrong and may or may not be fixable)
·
How to simulate data
SEDFIT-SEDPHAT - Global Analysis of Interacting
Systems by SV, SE, ITC, and SPR
Theme of this workshop is the characterization of interacting
systems with regard to the number, size, stoichiometry, and shape of reversible
macromolecular complexes, as well as the binding kinetics and thermodynamic
parameters governing the reaction. Tools are presented for the study of
self-associating systems, binary systems with single-site or multi-site
interactions, as well as cooperative multi-protein reactions with multiple mixed
species.
A big section of the workshop is dedicated to the behavior of
interacting systems in analytical ultracentrifugation. This includes the
analysis of sedimentation velocity on several levels of detail, by direct Lamm
equation fitting and/or isotherm analysis of weight-average s-values, boundary
populations and the application of Gilbert-Jenkins theory. It also includes
sedimentation equilibrium analysis and the generation of constraints via mass
conservation and suitable experimental design.
SEDPHAT provides functions for the global analysis of data
from other biophysical techniques, as well. Introductions to ITC and SPR are
given and tools for the analysis of their data are presented. Considerations
for the simultaneous modeling of data from different techniques in SEDPHAT are
introduced.
The workshop is a series of alternating lectures and guided
computer exercises by the participants on their own laptop. This workshop
assumes basic familiarity with SEDFIT (which may be acquired with the
self-learning tutorial prior to the workshop).
Preliminary Agenda:
·
Introduction to SEDPHAT
o
Principles of organization: global vs
local parameters, constraints, parameters in standard conditions vs
experimental conditions
o
Practical exercise transferring data between
SEDFIT and SEDPHAT, configurations, hybrid discrete/continuous model, links of
local parameters
·
Theory and practice of sedimentation
velocity of interacting systems
o
Global Lamm equation modeling, transport
method (weight-average s-values), constant bath theory and Gilbert Jenkins
theory, c(s)
o
Practical exercise of recognizing
interaction categories, direct Lamm equation fitting for reacting systems,
isotherm analysis of of sw(c), Gilbert-Jenkins isotherms, partial population
isotherms
·
Multi-signal sedimentation velocity for
multi-component systems
o
Theory and practical exercises
·
Introduction to ITC
o
Practical: importing data into SEDPHAT
o
Practical: global analysis of multiple
titrations for cooperative multi-protein complexes
·
Global sedimentation equilibrium analysis
for interacting systems
o
Theoretical overview, data analysis with
constraints from mass conservation, multi-signal acquisition, and from
experimental design strategies
o
Practical: sorting data, setting up SEDPHAT
global SE analyses, links, error analyses
·
On particle-solvent interactions in
analytical ultracentrifugation
o
effects of hydration and solvation in the interpretation of buoyant molar mass
and hydrodynamic friction
o
membrane proteins, nucleic acids,
denaturants, osmolytes
Introduction to SPR
o
EVILFIT for the calculation of affinity and
rate constant distributions
o
Direct binding and competition isotherm
analysis in SEDPHAT
·
SEDPHAT for fluorescence quenching and
fluorescence anisotropy analysis
Global fitting of binding data from
different biophysical techniques
·
Optimization and Statistical Error Analysis
o
Introduction to minimization routines, error
estimates by covariance matrix, Monte-Carlo simulations, F-statistics
projections, error analysis for distributions, regularization, Bayesian analysis
o
Practical exercises: determining a
confidence interval in sedimentation equilibrium analyses, scaling experimental
errors in multi-method fits.
·