Precision protein standard

The Th T stock solution was prepared by dispersing 8 mg of Th T in 10 mL of phosphate buffered saline (pH 7.0) containing 150 mM NaCl and stored in darkness before starting the measurements. The stock solution was diluted 50-fold in the same buffer on the day of analysis to generate the working solution. Aliquots (20 μL) of the control sample of fibrils and of NP-fibril composite were mixed with 5 mL of Th T working solution and allowed to stand for at least 1 min. The fluorescence spectra of the mixtures were measured using a fluorescence spectrophotometer (POLAR star Omega). The excitation wavelength was 440 nm (slit width = 10 nm) and the emission wavelength was 480 nm (slit width = 5 nm), with a scanning speed of 240 nm/min. Wavelength number was scanned from 190 nm to 270 nm. The fluorescence spectrum of the Th T working solution was deducted as background signal from the fluorescence spectra of the samples. SDS-PAGE was performed for molecular weight estimation of the samples, using a Bio-Rad instrument and precast Mini-protean 4–20% gradient gels. Samples were mixed with stain buffer, using Laemmli mixed with βMe (2-Mercaptoethanol) in 9:1 volume ratio, and then incubated at 90 °C for 10 min and quickly centrifuged for few seconds before starting the measurements. Protein ladder: Bio-Rad precision protein standard. Running buffer: Bio-Rad Tris/Glycine/SDS.

Approximately 3 μg of A. ceylanicum adult ESPs was mixed with 2× concentrated Laemmli-SDS buffer (Bio-Rad) with 5% 2-Mercaptoethanol (Sigma-Aldrich). The sample was heated at 95 °C for 5 min and 50 µL of protein sample was loaded into the wells of 12% Mini-PROTEAN TGX gel (Bio-Rad). The precision protein standard (Bio-Rad) was loaded into the first well at 10 µL without dilution with an SDS sample buffer. Electrophoresis was run for 50 min at 150 V using 1× Tris/Glycine buffer (Bio-Rad), [6 (link)]. The gel was fixed in (40% ethanol and 10% acetic acid), washed with distilled water, and stained with QC Colloidal Coomassie Stain (Bio-Rad). Gels were imaged in Azure Imaging Systems (C600, Azure Biosystems, Dublin, CA, USA) under LED visible fluorescence detection mode (data not shown).

For protein separation, samples were mixed with Laemmli sample buffer with or without 200 mM dithiothreitol (DTT), incubated for 5 min at 95°C and separated in a 3–15% polyacrylamide gel by discontinuous SDS-PAGE using a 3% stacking gel [17] (link). The Precision Protein Standard (Bio-Rad) was used as molecular marker. The separated proteins were Western-blotted onto polyvinylidene difluoride (PVDF) membranes (Immobilon-PSQ Transfer Membrane, Millipore) using a Trans-Blot SD semidry transfer cell (Bio-Rad) at 2.4 mA/cm² (MUC2) or 2.0 mA/cm² (AGR2) for 1 h with blotting buffer (Tris 48 mM, glycine 39 mM, SDS 1.3 mM and 10% or 20% methanol for MUC2 and AGR2 respectively). The membrane was blocked for 1 hour at RT in 5% non-fat milk in PBS with 0.1% Tween 20, and incubated 1 hour at RT or at 4°C overnight with the primary monoclonal antibodies anti-AGR2 (Abnova H00010551-M01, 1∶2000), anti-GFP (Sigma G6539, 1∶4000) or anti-Actin (Millipore MAB1501, 1∶5000) and subsequently with a secondary goat anti-mouse-HRP antibody (Southern Biotech 1∶2000). Membranes were developed with Immobilon Western chemiluminescent HRP substrate (Millipore), imaged using a LAS 4000 analyzer (Fujifilm), and the band intensities measured using the Multi Gauge v3.0 (Fujifilm) software. Band intensities of three replicated blots plotted as histograms using GraphPad Prism v6 (GraphPad, La Jolla, CA).