Immobiline drystrip gel

Immobiline dry strip gels with a linear pH gradient of 3–10 for isoelectric focusing were purchased from GE Healthcare Life Sciences (Rydalmere, NSW, Australia). Glycerol, thiourea, dichlorodiphenyltrichloroethane (DDT), and ampholyte at the corresponding pH range of 3–10 required to prepare the OFFGEL stock solution, were obtained from Agilent Technologies (Mulgrave, Victoria, Australia). ThT, acetonitrile (ACN), chloroform, TCEP, sulfuric acid (99.8% pure), and hydrogen peroxide (30% pure) were of analytical grade, obtained from Sigma-Aldrich (Castle Hill, NSW, Australia). Silicon wafers were obtained from MMRC Pty. Ltd. (Melbourne, Australia). The Chp F peptide with the sequence of DSGAQAAAAHSPGVLSGNVVQVPVHIPVNVCGNTIDVIGLLNPAFGNECEND5 (>95% purity) was synthesised by CS Bio Co (Menlo Park, CA, USA).

Protein precipitation of total extract was performed as in Buldain et al. (2016) [11 (link)]. In the case of the secretome, it was carried out at −20 °C in one volume of acetone plus 20% (p/v) trichloroacetic acid and 0.07% (v/v) 2-mercaptoethanol for 1 h. The sample was then centrifuged (11.400× g, 45 min, 4 °C), the supernatant discarded and the pellets washed three times with cold acetone, air-dried and resuspended in rehydration buffer (7 M urea, 2 M thiourea, 20 mM Tris, 4% (p/v) CHAPS, 0.5% (v/v) ampholyte, 20 mM DTT, 0.002% (p/v) bromophenol blue). Samples were sonicated using 2 pulses at an amplitude of 40 for 2 min, and stored at −20 °C until further use.
Afterwards, the isoelectric focusing (IEF) was carried out using 18 cm long Immobiline DryStrip gels (pH 3–10, GE Healthcare) and ReadyStripTM IPG Strips pH 3–6 (Bio-Rad), loaded with 400 µg of protein. All the two-dimensional electrophoresis (2DE) were made in triplicate and only the most informative gels of the two pH ranges used are shown in the results. The protocol described in Pellon et al. (2016) [14 (link)] was followed for the IEF and 2DE of the total extract, using 12.5% gels. For the secretome, an initial step of 150 V at 300 Vhr in the IEF protocol and the use of 13% polyacrylamide gels in the second dimension were the only modifications introduced.

Human retina protein isolated from 4 normal, 5 gliotic specimens, as well as 4 Müller glia cell preparations were combined to create 3 separate pools, each consisting of 150 μg of protein. 2D-DIGE analyses were performed as previously described (Heywood et al., 2011 ). Each protein pool was labelled with a different CyDye (600 pmol) as follows: Control retina was labelled with Cy3; gliotic retina with Cy5 and Müller glia with Cy2. Samples were then combined and added to Immobiline DryStrip gels (IPG) and run on an IPG Multiphor II Electrophoresis System (GE Healthcare, Little Chalfont, U.K.). For the second dimension, Isoelectric focusing (IEF) strips were re-equilibrated before resolving samples on an Ettan DALT twelve System separation tank using 12% acrylamide gels. Gels were fixed and imaged using a Typhoon scanner (Model 8600, GE Healthcare, UK). Spot comparison was performed using Progenesis Samespots software (Non-Linear Dynamics, Waters, UK). Gels were silver stained (Heywood et al., 2011 ) and protein spots excised, trypsin digested and processed as for mass spectrometry.

N-hydroxy succinimide ester Cy2, Cy3 and Cy5, urea, glycerol, mineral oil, immobiline DryStrip gels (18 cm, pH = 3–10, pH = 4–7 and pH = 6–11) and IPG buffer solutions (pH = 3–10, 4–7 and 6–11) were purchased from GE Healthcare (Piscataway, NJ). Acrylamide, DTT, Tris, glycine and SDS were purchased from Bio-Rad (Hercules, CA, USA). Dimethyl formamide (DMF), CHAPS, L-lysine, ammonium persulfate, iodoacetamide, agarose, bromophenol blue and TFA were purchased from Aldrich (Poole, Dorset, UK). Thiourea, TEMED, acetone, acetonitrile (ACN) and ethanol were purchased from Fluka (Buchs, Switzerland). Trypsin (sequencing grade) was purchased from Promega (Madison, WI). All buffers were prepared with Milli-Q water (Millipore, Belford, MA, USA). Imperial Protein Stain solution was purchased from Thermo Scientific (Rockford, IL, USA).

An aliquot of 5 mL from each peripheral blood sample was separated into serum fractions by centrifugation at 1000 × g for 10 min. Serum protein was harvested with the Albumin and IgG Removal Kit (Pierce, Thermo Scientific, Rockford, IL, USA) according to the manufacturer's instructions. The supernatant was subsequently collected and used for 2-D electrophoresis. Approximately 300 μg of protein was suspended in a rehydration solution (8 M urea, 2% CHAPS, 65 mM DTT, 0.2% pharmalyte [pH range 3–10] and 0.2% bromphenol blue) and applied to 18 cm pH 3–10 non-linear Immobiline^™ DryStrip Gels (GE Healthcare Bio-Sciences, Pittsburgh, PA, USA) for isoelectrofocusing [37 (link)]. The isoelectrofocusing was performed using an Ettan IPGphor^™ Instrument (GE Healthcare Bio-Sciences), and proteins in the IPG strips were subsequently placed on a 12% uniform SDS-polyacrylamide gel. The gels were silver-stained and scanned with an Image Scanner in transmission mode, after which the image analysis was undertaken using 2-D PDQuest (Bio-Rad, Hercules, CA, USA).