Abstract:
:Flow dialysis has found widespread use in determining the dissociation constant (KD) of a protein-ligand interaction or the amount of available binding sites (E0). This method has the potency to measure both these parameters in a single experiment and in this article a method to measure simultaneously the KD and E0 is presented, together with an extensive error analysis of the method. The flow-dialysis technique is experimentally simple to perform. However, a number of practical aspects of this method can have a large impact on the outcome of KD and E0. We have investigated all sources of significant systematic and random errors, using the interaction between mannitol and its transporter from Escherichia coli as a model. Monte Carlo simulations were found to be an excellent tool to assess the impact of these errors on the binding parameters and to define the experimental conditions that allow their most accurate estimation.
journal_name
Biophys Jjournal_title
Biophysical journalauthors
Veldhuis G,Vos EP,Broos J,Poolman B,Scheek RMdoi
10.1016/S0006-3495(04)74259-9subject
Has Abstractpub_date
2004-04-01 00:00:00pages
1959-68issue
4eissn
0006-3495issn
1542-0086pii
S0006-3495(04)74259-9journal_volume
86pub_type
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