prepared refolding buffer, consisting of 50 mM Tris-HCl pH 8.5, 1 M NaCl, 0.5 M L-arginine, 5 mM EDTA, 5 mM reduced glutathione and 1 mM oxidised glutathione. The pH was re- adjusted to 8.5 before addition of protein. The protein was left to refold without agitation for 6 days at 4uC then centrifuged at 30,0006g, 4uC for 30 min before overnight dialysis into 20 mM Tris-HCl pH 8.5. Soluble protein was harvested as supernatant after a second centrifugation and filtered (0.45 mM). Purification of Refolded MstnPP Dimer and Soluble Aggregates Purification of the in vitro refolded MstnPP dimer and misfolded soluble aggregates was performed by heparin affinity followed by gel filtration chromatography. The filtered protein solution was loaded onto a 5 mL HiTrap Heparin HP column (GE Healthcare) equilibrated with 50 mM Tris-HCl pH 8.5, at a flow rate of 1 mL/min. The flow-through containing unbound protein was discarded and a stepwise NaCl elution was used to elute the correctly folded dimer at 200 mM NaCl (H1) and the majority of soluble aggregates at 600 mM NaCl (H2). Elution was monitored by absorbance at 280 nm and differently folded forms were identified using non-reduced SDS-PAGE. Further purification of the MstnPP dimer was achieved using gel filtration chromatog- raphy. The 200 mM NaCl peak was filtered (0.22 mM), concen- trated to 350 mL and applied to a Superdex S200 10/300 HR column (GE Healthcare) equilibrated with 50 mM Tris-HCl, pH 8.5, 150 mM NaCl, at a flow rate of 0.5 mL/min. The dimer eluted at approximately 13.5 mL (PD) and was separated from any remaining soluble aggregates that eluted in the void volume (PA). Western Blotting Protein samples were separated on a 12% SDS-PAGE gel at 150 V and transferred to a nitrocellulose membrane overnight at 15 V (approximately 40–150 mA) in cooled Tris/glycine transfer buffer. The next day transfer of high molecular weight samples was ensured at 450 mA for one hour. Efficient transfer was confirmed by Ponceau S staining. Membranes were blocked with 5% non-fat milk in TBS-Tween (TBS-T; 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% Tween-20) for one hour at room temperature and subsequently incubated in rabbit anti-myostatin polyclonal primary antibody (Chemicon International), diluted 1/10,000 in 1% non-fat milk TBS-T, for 90 minutes. Following washing with TBS-T, membranes were incubated in a 1/50,000 dilution (1% non-fat milk, TBS-T) of horse radish peroxidase- conjugated anti-rabbit antibody (Jackson ImmunoResearch) for 90 minutes. All steps were performed at room temperature. After washing with TBS-T, proteins were visualised with the Super- Signal West Pico Chemiluminescent Substrate (Pierce) according to manufacturer’s instructions. Generation of MstnPP Amyloid Fibrils MstnPP soluble aggregates were sterile-filtered (0.22 mm), concentrated 10-fold and resuspended in dilute HCl solutions at a final concentration of 1 mg/mL, as measured by both Bradford assay and absorbance at 280 nm. Initially, a range of pH solutions were investigated for fibril formation from pH 1.6, as used for insulin amyloid formation [63] through to pH 6.3. Samples were placed at either 37uC or 60uC and monitored frequently for amyloid formation by Thioflavin T (ThT) fluorescence, TEM and CD spectroscopy. Subsequent to this optimisation, MstnPP aggregates were incubated at pH 5.3 (0.005 mM HCl) and 60uC for at least one week. Identical MstnPP amyloid fibrils were formed in equivalent solutions containing 0.1% sodium azide. Myostatin Amyloid Formation Generation of Insulin Protofibrils and Fibrils Insulin protofibrils and fibrils were produced as described [63]. In brief, different concentrations of insulin (bovine pancreas, Sigma) ranging from 0.5–2 mg/mL (85–340 mM) were prepared in 25 mM HCl (pH 1.6) and placed at 60uC for an hour (protofibrils) or at least overnight (fibrils) although incubation times were dependent on protein concentration. Negative-Stained Transmission Electron Microscopy Dimer and aggregate samples direct from purification (heparin affinity or gel filtration) were buffer-exchanged into water. Amyloid solutions were used directly in dilute HCl. 200-mesh carbon-coated Formvar grids were placed on 30 mL protein solution (0.5 mg/mL) droplets for 45 seconds. Excess sample was drawn off using filter paper and the grids were placed on an equal volume of 2% uranyl acetate for 45 seconds. Excess stain was drawn off as above and the grids were air-dried briefly before viewing with a Philips CM10 transmission electron microscope. Fibril measurement and statistical analysis was performed using iTEM software (Olympus) with a sample size of 10 fibrils from the same grid. Thioflavin T Binding Assays A solution of Thioflavin T (Sigma) was prepared at 400 uM in water [64] then diluted directly into the protein solution giving a final concentration of 20 mM per assay (as done previously for insulin [63]). A final protein concentration of 1 mg/mL was used for all samples. Experiments at pH 7.5 were performed after diluting both protein and ThT into 50 mM Tris-HCl pH 7.5, 100 mM NaCl. Fluorescence assays were conducted with the Perkin Elmer LS50B Luminescence Spectrometer with excitation at 450 nm and the emission spectrum measured from 460– 530 nm. Error bars are standard errors for four independent measurements. The Student’s t-test was performed using Graph- Pad Prism (GraphPad Software, Inc). Circular Dichroism Spectroscopy CD spectra in the far-UV region (180–260 nm) were obtained on a Chirascan CD spectrometer (Applied Biophysics) using a 0.1 mm path cell and protein concentrations of 1 mg/mL at 4uC. For all samples, 20 runs were performed with 1 nm readings taken every 2.5 seconds, followed by smoothing and baseline subtraction. For CD thermal denaturation, 1 nm/2.5 second readings were taken at every 5uC increase in temperature from 10–65uC with a 30 second equilibration time at each temperature and a tolerance level of 0.2uC. Protease Resistance Analysis MstnPP samples were tested for protease resistance using a myostatin to trypsin (bovine pancreas, Sigma) ratio of 100:1 or 20:1 (w/w). 100:1 solutions were incubated at 4uC, room temperature or 37uC. Samples were taken at 0.5, 1, 2, 3, 4 and 18 hours (overnight), immediately denatured to end the reaction and analysed by reducing and non-reducing SDS-PAGE. For subsequent protease resistance analysis of MstnPP amyloid fibrils, the myostatin to trypsin ratio was 20:1 (w/w) with incubation at 37uC for 4 and 18 hours and analysis by SDS-PAGE and TEM. Cell Culture and Incubation with Protein Aggregates C2C12 mouse myoblasts were cultured in Advanced Dulbecco’s Modified Eagle’s Medium (Gibco, Invitrogen) containing 4.5 g/L D-glucose and 110 mg/L sodium pyruvate and supplemented with 10% foetal calf serum, 4 mM L-glutamine and penicillin/ PLoS ONE | www.plosone.org 10 February 2010 | Volume 5 | Issue 2 | e9170