Workshop on Hydrodynamic and Thermodynamic Analysis
of Macromolecules with SEDFIT and SEDPHAT
April 12 - 16, 2010, Bethesda
This workshop will review strategies for hydrodynamic and thermodynamic analysis of sedimentation equilibrium and sedimentation velocity analytical ultracentrifugation (AUC), dynamic light scattering (DLS), isothermal titration calorimetry (ITC), fluorescence spectroscopy (anisotropy (FAI) and quenching isotherms), and optical biosensing (SPR) for the characterization of biological macromolecules in solution and their interactions.
The workshop will start on Monday morning and end on Friday afternoon. On the first day, we will provide an introduction into the experimental aspects of AUC, DLS and ITC. This part will take place in the laboratory. We will review the basic operation of the instruments, choice of buffers, and detection systems, and experimental design dependent on the system under study. For AUC, assembling the AUC cells and operating the XLA/I will be discussed in detail. The following days, will cover many advanced topics of theory, data analysis and experimental setup. These lectures are accompanied by practical computer exercises with the software. There will be dedicated one-on-one sessions for discussing each participant's data.
The workshop is aimed at researchers with at least limited experience in experimental biophysical methods. An integral part of the workshop is the presentation by the participants of their systems of interest and informal discussion of data.
Topics include:
Experimental
| Experiment design in AUC: choice of concentrations, buffers, optical systems |
| Conducting the AUC experiment: assembly of ultracentrifugal cells, adjustment of optics, operating the XLA/I |
| Operating the ITC and experimental design |
| Operating the DLS and experimental design |
Theory and Practical Data Analysis
| General aspects of non-linear regression and statistical error analysis |
| Overview of sedimentation velocity analysis with SEDFIT |
| Dynamic light scattering analysis in SEDFIT |
| Discrete species analysis by direct boundary modeling |
| Sedimentation coefficient and molar mass distributions |
| Interpretation of the goodness of fit, frictional ratio, and statistical analysis |
| Enhancing sensitivity and resolution by incorporating prior knowledge in c(s) with new Bayesian approach |
| Example: The detection of trace oligomeric components |
| Size-and-Shape distributions c(s,M) and c(s,f) |
| scale-free general sedimentation coefficient distribution c(s,*) |
| Overview of data organization in SEDPHAT, and exporting data from SEDFIT to SEDPHAT |
| Global analysis concepts with SEDPHAT |
| ITC analysis for single and multiple titrations |
| ITC analysis of ternary interactions |
| SPR analysis in EVILFIT: continuous distributions of rate and affinity constants |
| Sedimentation equilibrium analysis: global analysis of multi-wavelength data |
| Sedimentation equilibrium analysis with soft mass conservation models |
| General isotherm analysis with SEDPHAT |
| Hybrid discrete/continuous sedimentation coefficient distribution analysis |
| Analysis of self-associating systems by c(s), isotherms, and direct boundary modeling |
| Analysis of hetero-associating systems by sedimentation velocity |
| Introduction to analysis strategies based on Gilbert-Jenkins theory |
| Global modeling of the reaction boundary |
| Multi-signal sedimentation velocity analysis for heterogeneous interactions |
| Systematic noise elimination |
Format:
The first workshop day will have brief introductory lectures for the different methodologies. The practical aspects will be covered in a mixture of hands-on exercises and demonstrations. This will take place largely in our laboratories, alternating with review and discussion of experimental strategies in the seminar room. The following part of the workshop will be devoted to the practical data analysis and take place in a classroom. The goal is to ensure through hands-on training that the participants become familiar with the details of the use of the software, and confident with each type of data analysis. We will have a lecture series in the morning and early afternoon, to introduce and recapitulate the theoretical background as well as the concepts for their implementation in the software, mixed with practical exercises on examples, where each participant will conduct data analyses on his/her laptop concurrent with the step-by-step demonstration projected on the large screen.
From mid afternoon, the participants are expected to give short presentations on a topic of their interest. This is very informal. This can be, for example, a presentation of the biological background of a system of interacting proteins for which sedimentation analysis may be applied in the future; a presentation of an existing application of analytical ultracentrifugation; an introduction to another biophysical technique for studying macromolecules; the presentation of an open question regarding ultracentrifugation technique; the discussion of preliminary data; or simply the introduction of the speaker and her/his interests. The purpose of this section is to learn about areas of application and to stimulate the discussion. In the past, this section has become a favorite for most participants, since it allows to get to know each other's interests, and as a group to take advantage of unique expertise of each of the participants.
Preliminary List of Instructors:
| Andrea Balbo (NIBIB, NIH) |
| Chad Brautigam (Biochemistry Department, UT Southwestern Medical School, Dallas) |
| Patrick Brown (NIBIB, NIH) |
| Rodolfo Ghirlando (NIDDK, NIH) |
| Peter Schuck (NIBIB, NIH) |
| Huaying Zhao (NIBIB, NIH) |
This workshop takes place at NIH, Monday 8 am, to Friday 4 pm.
The number of participants is limited and Registration is required. The fee schedule can be found here. Each participant will need a laptop with SEDFIT and SEDPHAT and the example data installed. Installation instructions and other workshop preparations will be provided.
Additional questions regarding the program should be addressed to Peter Schuck (schuckp@mail.nih.gov). For questions regarding the organization, please contact Ms. Carline Coote.