quently, whether the binding of albumin and casein to this ECM proteins is upregulated when stretched. To conduct quantitative assays with sufﬁcient sensitivity, we chose to study ﬁbers made from the ECM protein ﬁbronectin (Fn) as a model system. Probing stretch-activated protein binding was made possible with a recently developed stretch assay that allows us to stabilize mechanically strained proteins in nonequilibrium structures and to read out their conformation optically with ﬂuorescence resonance energy transfer (FRET).9,10 To probe the adsorption and desorption kinetics as a function of the mechanical strain of ﬁbrillar Fn, casein and albumin (labeled with a third ﬂuorophore) were allowed to adsorb from solution. We further asked whether ﬁbrillar Fn could refold once albumin was bound to stretched ﬁbers and whether albumin would desorb once the force pulling on the ﬁbers was released. Finally, we probed whether bound albumin blocks access to speciﬁc binding sites, with a focus on probing the cryptic binding sites that regulate Fn-Fn ﬁbrillogenesis. FRET-Based Stretch Assay to Mechanically Tune the Conformation of Fibrillar Fibronectin. To tune the con- formation of Fn ﬁbers in a controllable manner, Fn ﬁbers were drawn manually from a concentrated Fn solution and deposited onto stretchable silicone sheets which were mounted onto a custom-built, uniaxial strain device.14 The ﬁbers parallel to the strain axis were stretched, whereas the vertically deposited ﬁbers were relaxed. Absolute ﬁber strains were deﬁned as 100(L-Lo)/Lo where L is the extended ﬁber length and Lo is the ﬁber length when fully relaxed (Klotzsch et al., submitted). To optically monitor conformational changes, in this large multimeric protein a small fraction of the plasma Fn was labeled with multiple donor and acceptor ﬂuorophores.9-11 Images were acquired, and the degree of FRET was measured, pixel-by-pixel, normalized by dividing the intensity of the acceptor channel by the intensity of the donor channel, hereafter referred to as IA/ID and visualized in false colors. Figure 1a shows that the stretching of Fn ﬁbers alters their conformation, and thus false color, where the relaxed Fn ﬁbers are red and the highly stretched ﬁbers are bluish-green. The histogram of the corresponding IA/ID ratios is shown in Figure 1b, indicating two major confor- mational populations representing the stretched and relaxed ﬁbers, respectively. Closer examination of each ﬁber indicates small pixel-to-pixel variations of Fn conformations. To calibrate the IA/ID ratios for every Fn batch, FRET-labeled Fn was also denatured in solution, where the onset of loss of secondary structure occurs at 1 M guanidine hydrochloride (GndHCl).9-11,14,28-29 We have shown previously that this IA/ID ratio equals the ratio seen for Fn ﬁbers strained 2-3 times their resting length10,14 and that the stretching of freely suspended ﬁbers beyond this mechanical strain causes a major increase in the exposure of cryptic sites buried in FnIII modules (Klotzsch et al., submitted). Stretching of Fn Fibers Enhances the Nonspeciﬁc Binding of Albumin and Casein. To test whether albumin and casein display up-regulated adhesion to Fn ﬁbers as a function of ﬁber extension, they were labeled with Alexa Fluor 633, a third, longer wavelength emitting ﬂuorophore. Nano Lett., Vol. 9, No. 12, 2009 When stretched or relaxed Fn ﬁbers were exposed to a 0.1 g/L solution of either labeled bovine serum albumin (BSA) or labeled casein, images of ﬂuorescent BSA or casein in Figure 1 show that a higher quantity of albumin (Figure 1a,c) and casein (Figure 1e,g) were bound to stretched (horizontal orientations) rather than to relaxed (vertical orientations) Fn ﬁbers (see also Figure S4, Supporting Information). The pixel intensities of BSA and casein appear brighter on stretched ﬁbers (Figure 1a). Since the number of Fn molecules per pixel volume changes as the ﬁbers get stretched, we normalized the intensity of the soluble protein (i.e., BSA or casein) by the intensity of the ﬂuorescently labeled ﬁbrillar Fn in each pixel. Pixel-by-pixel, the intensity of Alexa 633- labeled soluble protein (IAlexa633) was divided by the sum of Fn’s donor and acceptor intensities (ID + I ) using the A acquired FRET image with 488 nm excitation light. Similar results for normalized BSA and Fn binding were found when we simply used the intensity of the acceptor ﬂuorophore with direct acceptor excitation at 543 nm (see Figure S3, Sup- porting Information). Figure 1d,h shows the ratio of the Alexa 633 intensity of bound BSA and casein (Figure 1c,h, respectively) to the sum of IA + ID for all ﬁber pixels in the ﬁelds of view shown in Figure 1a,e. The data are shown as contour plots where the density of ratios is mapped to an artiﬁcial color code. The binding of both BSA and casein increases as Fn is progressively stretched. The transition from low binding to increased binding occurs when relaxed ﬁbers (0% strain) are stretched to about 100-140% strain. When the Fn ﬁbers are stretched further, albumin and casein binding levels plateau. It was not possible to determine which soluble protein had the maximum amount of binding at a given strain state since the ratio of Alexa 633 to Fn-DA IA + ID depends upon the labeling ratio of BSA and casein. However, the relative increase in binding was even more pronounced with casein (Figure 1h). While this ﬁrst set of experiments was conducted under physiological buffer conditions, the experiment was repeated in the presence of 10% fetal calf serum. We labeled all proteins in whole bovine serum with Alexa Fluor 633 and repeated the colocalization binding experiments described above (Figure S4, Supporting Information). Again, we observed an increased albumin binding as the ﬁbers were stretched, demonstrating that the increased binding behavior to mechanically stretched Fn ﬁbers is physiologically rel- evant. While the results were similar to albumin and casein in that a signiﬁcant increase in serum binding was observed up to a 100-140% absolute strain (Figure S4, Supporting Information), beyond this point, no signiﬁcant increase in binding was detected. As a negative control of a binding site that is exposed under equilibrium, we also investigated how the L8 mono- clonal antibody, which binds only within residues 526-675 spanning FnI9 and FnIII1,30 adheres to stretched versus relaxed Fn following the same colocalization procedure. Contrary to the results from all other proteins examined in this report, the L8 antibody showed decreased binding to stretched Fn ﬁbers (Figure 1i-j). L8 binding to Fn ﬁbers 4159