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Figure 3
Binding of HNF4α-DBD to duplex 21-mer target DNA detected by EMSA. Reactions were carried out in 10 mM Tris (pH 8.0 at 293 K), 1 mM EDTA, 100 mM NaCl, 1 mM MgCl2, 1 mM DTT, 4% glycerol, 100 µg ml−1 BSA. (a) Forward titration. All samples contained 0.21 µM duplex 21-mer. Samples contained HNF4α-DBD protein, from the second lane, at 0.41, 0.82, 1.23, 2.05, 2.87, 3.69, 4.51, 5.33, 6.56, 8.2, 12.0 and 20.5 µM, respectively. (b) Serial dilution. The first lane contained reference DNA; the initial sample (second lane) contained 0.2 µM duplex 21-mer and 17.9 µM HNF4α-DBD protein. Each succeeding lane contained an aliquot of the previous sample diluted 1.2-fold. (c) Determination of stoichiometry and association constant. Graph of ln[PnD]/[D] as a function of ln[P]. The slope about the mid-point of the reaction (where ln[PnD]/[D] = 0) gives the stoichiometry (n = 2.3 ± 0.2). The data used for this determination are indicated by filled circles. The data near binding saturation (filled squares) deviate from the linear relationship and were excluded from the fit. A fit of (1)[link] to the linear data returned an intercept value of ln[P] = −12.58 ± 0.03, equivalent to Kobs = 8.48 ± 0.67 × 1010M−2.

ISSN: 2053-230X
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