(5 µM) on the pre-treated surface.

<p>The curves, (generated as described in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044287#pone-0044287-g006" target="_blank">Figure 6</a>) were fit to: <b>A</b>: a single exponential dissociation model (red line) or <b>B</b>: a single association model (red line). Calculated rate constants from these fits gave the following values: k_a = 3.7 10⁶ M⁻¹s⁻¹; k_d = 1.8 10⁻³s⁻¹; K_D = 4.9 10⁻¹⁰ M.</p

SPRi kinetic curves of PFV-1 IN (200 nM) interacting with immobilized dsDNA on the pre-treated surface.

<p>The values for % reflectivity were obtained from direct CCD camera measurements averaged across each spot shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044287#pone-0044287-g005" target="_blank">Figure 5</a> and as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044287#pone.0044287-Bouffartigues2" target="_blank">[27]</a>. <b>A</b>: Changes in % reflectivity at selected spots on the SPRi surface as a function of time as PFV-1 IN passes over the prism surface. The curves show binding to spots containing different concentrations of DNA (1, 2.5, 5 and 10 µM) or to reference spots (containing no DNA) outside of the zone to which DNA was applied (designated as background). <b>B</b>: The kinetic curve after subtraction of the background for reaction taking place on a spot where 10 µM DNA solution was deposited. The red line is a fit carried out by applying a single exponential model where is the % reflectivity at time <i>t</i>; is the amplitude of the phase, and the observed rate constant , is the association rate constant, is the dissociation rate constant calculated from a simple exponential fit of the dissociation phase using and [<i>C</i>] is the concentration of PFV-1 IN (200 nM). C: same curve as shown in <b>B</b> but fitted (red line) with a double exponential model for both association and dissociation. The model for association is and dissociation is obtained from where is the % reflectivity at time <i>t</i>; and and are the respective dissociation rate constants for the two phases and ; and .</p

Density of DNA adsorbed on a pretreatedEG4 SAM as a function of the applied SH-5T-dsDNA concentration.

<p>The incubation time of the EG4 SAM is 30 s. The error bars correspond to the dispersion of the results over 4 separate measurements.</p

SPRi difference images of the biochip surface at different times during the course of the experiment.

<p>The EG4 adsorption time was 30 s. 4 different SH-5T-dsDNA solution concentrations were spotted in rows from right to left: 10, 5, 2.5 and 1 µM respectively. Each SH-5T-dsDNA concentration was spotted 3 times in the same column. <b>A</b>: t = 0 s, no PFV-1 IN protein injected. <b>B</b>, <b>C</b>, and <b>D</b> are images taken at t = 18 s, t = 3 min and t = 5 min respectively after 200 nM PFV-1 IN injection. <b>E</b> is the image taken 12 min after the injection was stopped. <b>F</b>, <b>G</b> and <b>H</b> are images taken at t = 9 s, t = 2 min, t = 6 min and t = 10 min after the start of the 0.1% SDS injection.</p

Calculated rate constants from fits of the type shown in Figure 5C.

<p>Values are shown based on binding to the four DNA concentrations spotted on the surface; errors refer to standard error for the rate constants calculated for each DNA concentration. The equilibrium dissociation constants K_D1 and K_D2 were calculated from the ratio of the respective kd/ka values.</p

SPRi difference images of PFV-1 IN interacting with the biochip surface.

<p>ssDNA solutions at 50, 25, 10, 6, 3, 1, 0.5 and 0.1 µM were spotted in replicates on the pre-treated surface. The entire spot image was between 0.8 and 0.9 cm in diameter and each individual spot was between 400 and 450 µm in diameter. The spotting, of decreasing ssDNA concentration, starts from left to right, and downward. <b>A</b>: ssDNA spotted on EG4 pre-treated surfaces, PFV-1 IN at 200 nM was flowed across the SPRi surfaces containing the spotted ssDNA molecules. Difference images during the injection are shown in panels <b>a</b> and <b>b</b>, while panel <b>c</b> is a difference image after the injection of the protein was stopped. Panel <b>d</b> and <b>e</b> shows difference images during the injection of a solution of 0.1% SDS across the surface during the dissociation phase of PFV-1 IN. Panel <b>f</b> shows difference images of PFV-1 IN retained at the surface after the SDS injection. <b>B</b>: Surfaces were prepared as previously by spotting ssDNA at various concentrations on the pre-treated surface. Then the complementary ssDNA oligomers was flowed across the surface in order to hybridize and form dsDNA before injecting PFV-1 IN at 200 nM across the SPRi surface. Panels <b>a</b> to <b>c</b> are difference images of PFV-1 IN flowing across the SPRi surface and panel d is a difference image after the injection of the protein was stopped. Panel <b>e</b> and <b>f</b> are difference images during and after the injection of a 0.1% SDS solution.</p

Cartoon describing the model for PFV-1 IN binding to DNA and subsequent accretion of proteins.

<p><b>A</b>: PFV-1 IN binds to the DNA; <b>B</b>: the nucleoprotein complex then binds more protein; <b>C</b>: which in turn continues to oligomerise until, <b>D</b>: the protein completely covers the DNA. The molecules are not to scale nor do they depict actual known orientations. The rate and equilibrium constants associated with B and C may be interchanged, as the model does not allow attribution of either to the structural forms suggested. The PFV-1 IN is represented in the monomeric form for simplicity.</p

Densities of DNA immobilized on pre-treated surfaces.

<p>The amount of DNA retained at the surfaces was calculated as described in the experimental section. <b>A</b>: Density of dsDNA with (OH-5T-dsDNA) and without (OH-dsDNA) the T₍₅₎ spacer on pre-treated surface. <b>B</b>: Density of the thiolated dsDNA with (SH-5T-dsDNA) and without (SH-dsDNA) the T₍₅₎ spacer on pre-treated surface. <b>C</b>: Density of immobilized DNA on the pre-treated gold surface as a function of the EG4 SAM immersion time ranging from 30 s to 2 hrs. SAM adsorption time = 0 corresponds to the bare gold surface. The SH*-5T-dsDNA and C*-dsDNA indicates which of the DNA strands were ³²P radiolabelled, namely the thiolated and the complementary strand respectively. OH-dsDNA refers to a ³²P radiolabelled non-thiolated dsDNA. All the dsDNA strands have the same sequence. <b>D</b>: Relative amount of double stranded DNA adsorbed on the surface after the pre-treated gold surface was immersed in 10 µM dsDNA solutions. The % dsDNA adsorbed is calculated from the ratio between C*-dsDNA and SH*-5T-dsDNA, 100% corresponding to the SH*-5T-dsDNA density. The error bars correspond to the dispersion after accumulating 8 experiments for each EG4 adsorption time.</p

Difference images obtained from SPRi of antibodies binding to GAD65 immobilised at a gold/GLISS surface.

<p>Spots (400 µm diameter) containing GAD65 protein were immobilised by spotting at the surface using Hamilton Starlet equipped with a PinTool tip and modified software. GAD1 antibody (4.8 nM in 200 µl PBS) was flowed across the surface at 20 µl/min as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012152#s2" target="_blank">Methods</a> and differential images obtained using the GenOptics SPRi device. The red circle illustrates the area that is generally chosen to calculate the pixel density at any given time in order to generate curves of the type shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012152#pone-0012152-g003" target="_blank">Figure 3</a>.</p

Differential binding to SPRi surfaces.

<p>Two independent surfaces were created either using classical SAM constructed from undecanoic acid as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012152#pone.0012152-Bouffartigues1" target="_blank">[2]</a>, or the GLISS protocol described here. Serum (stock concentration 60mg/ml diluted to a final concentration of 0.6 mg/ml) in PBS was passed across the surfaces at 25 µl/min. The three images for each surface refer to images prior to, during and after injection of the serum. A) Differential images of classical undecanoic based SAM surfaces B) Differential images of GLISS prepared surfaces C) Changes in % reflectivity as a function of time derived from images of the type shown in A and B.</p