XMD8-92 Supplier br Materials and methods br Acknowledgments

Materials and methods
Acknowledgments We would like to thank all members of the Imhof and Heun group for critical discussions and feedback and the anonymous reviewer for valuable comments and suggestions to improve the manuscript. Work in the lab of A.I. was supported by grants from the German Research Council (Deutsche Forschungsgemeinschaft (DFG; Projekt IM 23/9-1)) and the European Union (EpiGeneSys, 257082). Work in the lab of P.H. was sponsored by an ERC (ERC-2012-StG_20111109) grant (ERC-BioSynCen).
Data In the data we included size exclusion chromatography data of soluble eADF4(C16) in a combination with light scattering revealing that the protein is monomeric before assembly (Fig. 1). A defined structural state is important before starting any kinetic study, since assemblies may significantly influence the kinetics by accelerating the nucleation (Fig. 2). The assembled mature fibrils were visualized by transmission XMD8-92 Supplier microscopy (TEM) (Fig. 3) and atomic force microscopy (AFM) (Fig. 4), revealing fibrils typically 10nm in diameter and 1µm in lengths. Sonication led to significantly shorter fibrils, as shown by AFM, increasing the number of the active fibril ends. Sonicated fibrils can be used as seeds to by-pass nucleation in eADF4(C16) assembly (Fig. 5). We further show sedimentation kinetics of spider silk in the presence of different NaCl concentrations revealing very slow protein aggregation (Fig. 6) in comparison to the fast assembly triggered by phosphate ions [1], which indicates that ion masking events are less important for the protein-protein interation during spider silk assembly.
Material and methods
[describe in 3–5 bulleted points why this data is of value to the scientific community] Experimental design, materials and methods As described in ‘Determination of Supplier-to-Supplier and Lot-to-Lot Variability in Glycation of Recombinant Human Serum Albumin Expressed in Oryza sativa’ [1,2,3,5].
Materials Chemicals, essentially FA-free pHSA (A3872 Lot#090M7001V, ≥99% purity), recombinant human serum albumin expressed in Saccharomyces cerevisiae (ScrHSA, Lot# SLBD2407, ≥99% purity, Albucult), O. sativa [OsrHSA, Lot# SLBC7527V (OsrHSA-sig-C), Lot# SLBG7405V (OsrHSA-sig-G), Lot# SLBH9636V (OsrHSA-sig-H) and Lot# SLBJ1196V (OsrHSA-sig-J) 100% purity, Cellastim] and Pichia pastoris (PprHSA, Lot# 080M1580V, ≥99% purity, Albagen) were sourced from Sigma-Aldrich (St. Louis, MO, USA). OsrHSA was also obtained from eEnzyme LLC (Gaithersburg, MD, USA) (OsrHSA-phy) (Lot# 20130110, >99% purity, Phyto-HSA), ScienCell Research Laboratories (Carlsbad, CA, USA) (OsrHSA-sci) (Lot# BJABAA42, ≥99% purity, Oryzogen) and amsbio LLC (Cambridge, MA, USA) (OsrHSA-ams) (Lot #20101008, >95% purity, ecoHSA). Recombumin (Lot# PDP100106) was donated by Novozymes Biopharma (Cambridge, MA, USA). Amicon Ultra 0.5ml 3000Da molecular weight cut-off (MWCO) centrifugal filter devices were purchased from Millipore (Billerica, MA, USA). Trypsin and chymotrypsin were Promega sequencing grade purchased from Fisher Scientific Canada (Ottawa, ON, Canada). Vivacon 500 10kDa molecular weight cut-off filters were from Sartorius Stadium Biotech North America (Bohemia, NY, USA).
Albumin sample preparation Albumin samples were prepared as described previously [4]. Briefly, samples were buffer exchanged into 5mM sodium phosphate (pH 7.4), with Amicon Ultra 0.5ml 3000Da MWCO centrifugal filter devices after pre-rinsing the filters with buffer. Protein concentrations were measured using a bulbourethral glands BCA assay kit (Sigma-Aldrich, St. Louis, MO, USA). Protein integrity after buffer exchange was assessed with 1-D SDS−PAGE using SYPRO Ruby protein stain (Molecular Probes, Eugene, OR, USA) and a Bio-Rad Molecular Imager Gel Doc XR+ system with Quantity One 1-D analysis software according to the manufacturer’s instructions (Bio-Rad, Mississauga, ON, Canada).
Liquid chromatography–mass spectrometry (LC–MS) analysis—Sample preparation