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Using In Vitro Fluorescence Resonance Energy Transfer to Study the Dynamics Of Protein Complexes at a Millisecond Time Scale
Chapters
- 00:04Title
- 00:33Designing the FRET Assay
- 01:12Preparation of Cul1AMC•Rbx1, the FRET Donor Protein
- 04:42Preparation of FlAsHCand1, the FRET Acceptor Protein
- 05:18Testing and Confirmation of the FRET Assay
- 06:08Measuring the Association Rate Constant
- 07:37Measuring the Dissociation Rate Constant of Cul1•Cand1 in the Presence of Skp•F-box Protein
- 08:33Results: Dynamics Of Protein Complexes
- 10:30Conclusion
Protein-protein interactions are critical for biological systems, and studies of the binding kinetics provide insights into the dynamics and function of protein complexes. We describe a method that quantifies the kinetic parameters of a protein complex using fluorescence resonance energy transfer and the stopped-flow technique.
