Table 1 Advantages and disadvantages of techniques that study DNA-binding proteins
From: DNA–protein interaction studies: a historical and comparative analysis
Technique [References] | Technique | Applications | Case studies | |
|---|---|---|---|---|
Pros | Cons | |||
Inexpensive and easy | The interaction may not withstand the filtration process Impossible to recover the resulting products | Identify nucleic acid–protein interactions | Prabu et al. [45] | |
Fast and easy Powerful and sensitive Semi-quantitative | Identify nucleic acid–protein interactions Identify complexes in a cell/tissue extract | Liu et al. [46] | ||
UV cross-linking is not invasive and does not practically disturb the molecular structures Laser cross-linking is simple and fast and decreases the probability of damaging the molecular structures | Formaldehyde, glutaraldehyde and UV standard cross-linking methods are non-specific | Identifies the molecules that participate in a DNA–protein complex, even though some of them may not be directly in contact with the DNA | ||
Immunoblotting analysis combined with EMSA performs only one diffusion blotting Electrophoretic ‘supershift’ assay only uses one gel and does not need any diffusion blotting All variants are relatively fast and easy | Electrophoretic ‘supershift’ assay requires the purification of the antibody preparation Shift-Western Blotting involves two blotting membranes | Detect if a certain protein is present in a nucleic acid–protein complex (electrophoretic ‘supershift’ assay) and estimate its size (shift-Western Blotting and immunoblotting analysis combined with EMSA) Identify the nucleic acid–binding proteins that link to a certain nucleic acid and estimate their molecular weight (2D electrophoresis (EMSA + SDS-PAGE) and South-Western Blotting) | Wang et al. [47] | |
EMSA and in vitro binding with a cell extract using mutated probes are quick and easy Y1H and PTA provide reliable results and are relatively direct and sensitive Y1H is quick | PTA takes a long time to be performed and involves several steps | Determine if a certain TF binds to a given sequence in vivo | Liu et al. [46] | |