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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.

 

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