Plusone repel silane es

To prepare p14^ARF-NPM1 and p14^ARFΔH1-3-NPM1 condensates for fluorescence microscopy analysis, the recombinant p14^ARF proteins (p14^ARF and p14^ARFΔH1-3) were resuspended from lyophilized powders using 100% dimethyl sulfoxide (DMSO) and added directly to solutions of NPM1, at room temperature, such that the final NPM1 concentrations were 10 μM. The final buffer contained 10 mM Tris pH 7.5, 150 mM NaCl, 2 mM DTT, 1.67% DMSO. Condensate suspensions were incubated for 1 hr at room temperature before being transferred to 16-well CultureWell chambered slides (Grace BioLabs, Bend,OR,USA) pre-coated with PlusOne Repel Silane ES (GE Healthcare, Pittsburgh, PA, USA) and Pluronic F-127 (Sigma- Aldrich, St. Louis, MO, USA). Images were acquired on a 3i Marianas spinning disk confocal microscope (Intelligent Imaging Innovations Inc., Denver, CO, USA) using a 100x oil immersion objective (N.A. 1.4).

Sodium hyaluronate (100–150 kDa) was purchased from Lifecore Biomedical. Dialysis membranes Spectra/Por (MW cut off 3.5 kDa) were purchased from SpectrumLabs. Tetrahydrofuran dry, hydrochloric acid (HCl) 37%, Dulbelco’s phosphate buffer saline (DPBS) and sodium chloride were purchased from Fischer Scientific. 3-(Trimethoxysilyl)propyl methacrylate, N-Boc-ethylenediamine, triethylamine anhydrous, acryloyl chloride, dioxane, N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride, 1-hydroxybenzotriazole hydrate, acetonitrile anhydrous, sodium hydrogen carbonate and photo-initiator Irgacure 2959 were purchased from Sigma Aldrich. Calcein AM, propidium iodide, minimum essential medium (MEM) without phenol red, Dulbecco’s modified Eagle medium (DMEM), high glucose Glutamax and fetal bovine serum (FBS) were purchased from Fisher Scientific. RGDSC, 5FAM-RGDSC, GCGYRGDSPG and 5FAM-GCGYRGDSPG peptides were purchased from Innovagen AB. Glass coverslips and absolute ethanol were purchased from VWR. Plus One Repel-Silane ES was purchased from GE Healthcare Life Sciences.

Microscopy plates (Greiner Bio, Kremsmünster, Austria) and slides (Grace BioLabs, Bend, OR, USA) were coated with PlusOne Repel Silane ES (GE Healthcare, Pittsburgh, PA, USA) and Pluronic F-127 (Sigma-Aldrich, St. Louis, MO, USA) and washed with water before the transfer of protein solutions. Fluorescent microscopy experiments were performed using a 3i Marianas spinning disk confocal microscope (Intelligent Imaging Innovations Inc., Denver, CO, USA) with a 100X oil immersion objective (N.A. 1.4). The phase diagram depicted in Figure 2d was generated by computing the average of the index of dispersion of fluorescent microscopy images of 5 images per well. The threshold for positive phase separation has been set to 10% of the maximum value. Fluorescent recovery after photobleaching (FRAP) experiments were performed by bleaching a circular area with a diameter of 1 μm in the center of droplets (n=12) to approximately 50% of initial fluorescence intensity. The observed fluorescence intensities were then normalized to global photobleaching during data acquisition and fitted as a group to determine recovery times according to ^{111 (link)} $$I_t=\frac{I_0+I_{\mathrm{\infty }}\frac{t}{t_{\raisebox{1ex}{$1$}\!\left/ \!\raisebox{-1ex}{$2$}\right.}}}{1+\frac{t}{t_{\raisebox{1ex}{$1$}\!\left/ \!\raisebox{-1ex}{$2$}\right.}}}$$
Uncertainty in the reported half-lives represent the standard error of the fit data. FRAP experiments were performed one hour after the mixing of components.