Mfp 3d microscope

AFM measurements were performed using a G5¹–G5⁷ construct with two cysteine residues incorporated at the C terminus, to allow attachment to the gold-covered surfaces via gold-thiol covalent attachment. All AFM measurements were carried out in 20 mM Tris (pH 7.5), 150 mM NaCl, at 25 °C, using an Asylum Research MFP-3D microscope. Silicon nitride cantilevers with nominal spring constant of 30 pN nm⁻¹ (Bruker MLCT) were used and calibrated using the thermal method68 . One hundred-microlitre protein solution (250 μg ml⁻¹ in AFM buffer) was adsorbed onto a gold surface and the AFM cantilever tip was used to pick it up by nonspecific adhesion, and then retracted at a constant speed (200, 800, 1,500, 3,000 and 5,000 nm s⁻¹), measuring the force exerted by the protein in the process. Three independent experiments (different cantilevers and surfaces) were performed for each pulling speed. The unfolding force for all events from acceptable traces were measured and their force-extension profiles fitted to the worm-like chain model69 (with the persistence length fixed to 400 pm) using the IGOR Pro 6 software (WaveMetrics) to obtain ΔL_c values. The data from triplicates were pooled and the force and ΔL_c probability histograms were generated for each retraction rate. The modal force and ΔL_c values were calculated from Gaussian fits to the histograms using Mathematica 10 (Wolfram Research).