Nonisotopic quantitative analysis of protein-DNA interactions at equilibrium.
Two versions of an enzyme-linked immunosorbent assay-type method to quantify protein-DNA interactions at equilibrium were developed. The first variant comprised immobilization of DNA-binding protein on microtiter plates, incubation with biotinylated DNA, and tagging of bound DNA with streptavidin- and biotin-substituted horseradish peroxidase. In the second version, biotinylated DNA was immobilized on streptavidin-substituted microtiter plates, incubated with DNA-binding protein, and bound protein was quantified with specific antibodies. To illustrate the method, the interaction of a fusion protein between glutathione-S-transferase and the DNA-binding domain of the helicase-like transcription factor with its cis-element (the B box of the plasminogen activator inhibitor-1 promoter) was determined with both versions: a 1:1 stoichiometric interaction with an equilibrium dissociation constant (Kd) of 1 nM was found, which is similar to the value determined by electrophoretic mobility shift assay, demonstrating the validity of the assays.
Benotmane
A M
AM
Center for Molecular and Vascular Biology, University of Leuven, Belgium.
Hoylaerts
M F
MF
Collen
D
D
Belayew
A
A
D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D001710:Biotin / Q000737:chemistry / Q000378:metabolism; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005982:Glutathione Transferase / Q000737:chemistry / Q000378:metabolism; D006735:Horseradish Peroxidase / Q000737:chemistry / Q000378:metabolism; D019809:Streptavidin
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D001710:Biotin; D004247:DNA; D019809:Streptavidin; D006735:Horseradish Peroxidase; D005982:Glutathione Transferase