Ac160ts silicon cantilevers

Human recombinant YB-1 was expressed in Escherichia coli and purified as previously described (26 (link)). Purified protein was dialyzed against 200 mM NaCl, 20 mM HEPES–KOH pH 7.6, 1 mM Dithiothreitol (DTT) and stored at −80°C. Human recombinant TIA-1 was purchased from Novus Biological and we controlled that the protein can bind and also aggregate mRNA in vitro. Interactions between TIA-1, YB-1 and mRNA were probed in 20 mM 2-(N-morpholino)ethanesulfonic acid (MES)–KOH, pH 6.8, 1 mM ethylene glycol tetraacetic acid (EGTA), 50 mM KCl, 2 mM MgCl₂ and 10 μM spermidine. Ten microliters of each sample were deposited on freshly cleaved mica and dried for AFM imaging as described previously (31 (link)). All AFM experiments were performed in intermittent mode with a multimode AFM instrument (Digital Instruments, Veeco, Santa Barbara, CA, USA) operating with a Nanoscope IIIa controller. We used AC160TS silicon cantilevers (Olympus, Hamburg, Germany) with resonance frequencies of ∼300 kHz. The applied force was minimized as much as possible to reduce sample deformation. Images were collected at a scan frequency of 1.5 Hz.

Ten microliters of each sample were deposited on freshly cleaved mica and dried with a filter paper for AFM imaging. The electrostatic adsorption of both protein:mRNA or protein:DNA complexes on mica was mediated by divalent magnesium cations which are present in the binding buffer (20 mM Hepes [pH 7.6], 50 mM KCl, 4 mM MgCl₂). All AFM experiments were performed in intermittent contact mode with a multimode AFM instrument (Digital Instruments, Veeco, Santa Barbara, CA, USA) operating with a Nanoscope IIIa controller. We used AC160TS silicon cantilevers (Olympus, Hamburg, Germany) with resonance frequencies of around 300 kHz. The applied force was minimized as much as possible. Images were collected at a scan frequency of 1.5 Hz and a resolution of 512 × 512 pixels. The scan frequency was typically 1 Hz per line, and the modulation amplitude was of about a few nanometers. Statistical analyses were performed using the nanoScope analysis software.

Microfibrillated cellulose (MFC) with a nominal fiber width of 50 nm and several hundred micrometers of length was purchased from Maine University (3.0 wt % aqueous gel) [58 ]. Further characterizations and information about the MFC can be found in [58 ]. Functionalized multiwalled carbon nanotubes (MWCNTs) with hydroxy and carboxyl groups (–OH and –COOH), outer diameter between 20 and 50 nm and an average length of 5 μm were produced at CTNano/UFMG [59 (link)–61 (link)]. Morphological analysis was carried out by scanning electron microscopy (SEM) in a Quanta 200 FEG, using secondary electrons between 2 and 10 kV. Atomic force microscopy (AFM) was carried out on a Bruker MultiMode8 SPM using the intermittent contact mode. AC160TS silicon cantilevers from Olympus with a typical spring constant of k ≈ 46 N/m, a nominal radius of curvature of r ≈ 7 nm, and a resonant frequency of ω₀ ≈ 300 kHz were employed. Heat flow and weight changes of selected solvents were determined by thermogravimetric analysis (TGA) using a PerkinElmer STA 8000 device. Electrical measurements were performed using a lock-in amplifier (SR830 DSP Stanford Research Systems), a pre-amplifier (model 1211 DL instruments), and a multimeter (model 2000 Keithley), which were controlled by a computer.