Table 3 Advantages and disadvantages of techniques that describe DNA-binding reactions
From: DNA–protein interaction studies: a historical and comparative analysis
| Technique [References] | Technique | Applications | Case studies | |
|---|---|---|---|---|
| Pros | Cons | |||
| Filter binding assay and EMSA [3, 20, 62] |
The Michaelis–Menten analysis can be applied use of intermediate DNA concentrations enables to determine directly and accurately the equilibrium constant The use of high DNA concentrations enables to determine the stoichiometry CE-LIF enables instant on-column visualisation, automated operation and computerised data analysis CE-LIF has small sample requirements, is highly sensitive and presents rapid analysis times |
Need to use purified protein and quantify its concentration The use of low DNA concentrations assumes 1:1 stoichiometry Need to perform serial dilutions to determine high affinities |
Compare binding affinities CE-LIF enables DNA–protein complex and DNA quantification and stoichiometry determination CE-LIF enables simultaneous measurements of electrophoretic mobility and fluorescence anisotropy | Prabu et al. [45] |
| SPR [42] |
Very sensitive, fast and easy Real-time assay More adequate than EMSA when comparing wild-type and mutant proteins | If more than one protein bind cooperatively, the results can be misleading | Measure binding affinities and kinetics directly and simultaneously | Song et al. [74] |