Figure 2. Force-induced exposure of cryptic cysteines on FnIII7 and FnIII15 evaluated with iodoacetamide and bis-ANS binding to Fn ﬁbers physisorbed to silicone sheets. (a) Fibers were pulled from a drop of wild-type Fn (blue) and deposited onto a silicone sheet that was mounted into a uniaxial stretch device.14 As a negative control, a drop of alkylated Fn (red) randomly labeled with Alexa 633 on lysines was positioned at the opposite side, and ﬁbers were pulled and deposited in between ﬁbers of alkylated Fn. (b) Differential interference contrast image of prepared ﬁber sample after it had been washed and stretched in BPS buffer containing 2% BSA. (c) Fluorescence emission of Alexa 633 conjugated to 5% of the ﬁbrillar Fn molecules. (d) Alexa 488 maleimide binding to stretch-exposed cryptic binding sites. After stretching, 5 ng of Alexa 488 maleimide were added to the solution and allowed to react for 15 min. (e-i) Differentially strained Fn ﬁbers that were deposited under various angles to the strain axis. After deposition, all exposed hydrophobic patches on these ﬁbers were blocked with iodoacetamide. Data analysis was performed by calculating the Alexa 488 to Fn-633 ratios. (j-k) Stretch-dependent binding of bis-ANS to Fn ﬁbers. Fn ﬁbers containing 5% Alexa Fluor 633- labeled Fn (Fn-633) were manually deposited onto prestretched silicon sheets in various orientations and subjected to a 0 and a 300% absolute strain, as indicated. Bis-ANS was then adsorbed for 45 min to the substrates, and the ratio of bis-ANS over the Fn-633 intensity was calculated for each pixel ((j) inset histogram). The intensity ratio was converted into a false shade of blues, where lighter colors indicated a greater degree of bis-ANS binding, and was displayed in the spatially resolved representative image shown. (k) The intensity ratio of the bix-ANS over the Fn-633 ﬂuorescence, computed pixel-by-pixel, for 48 ﬁbers oriented in various directions versus their absolute strain. Scale bar in (b) is 100 µ m. 4162 Nano Lett., Vol. 9, No. 12, 2009