Glycerol

The protocol used was similar to that previously described by our group^{1 (link)}, but we introduced the modifications described below.
We incubated 40 µL of CSF and serum samples with 5 µL of 0.5 M dithiothreitol and 5 µL of 1 M Tris/HCl pH 9.7, for 45 min.
For protein separation, we used an agarose gel consisting of 0.3 g of agarose (GE Healthcare); 3.6 g of sorbitol (Sigma-Aldrich); 1.25 mL of each Pharmalyte, pH 4–6.5 and pH 3–10 (GE Healthcare); 2.5 mL of glycerol, and 22.5 mL of water.
To detect IgA, we used a biotinylated anti-human IgA antibody (Jakson Immunoresearch) diluted (1:20.000) in the blocking solution. After incubating the membrane overnight at 4 °C, the labeling was developed using streptavidin-HRP (Jakson Immunoresearch) diluted (1:1.000) in blocking solution.
To study the sensitivity of this new assay (IEF and immunodetection), we analyzed paired CSF and serum samples from 3 MS patients showing OGIgAB. We applied 5 µL of twofold serial diluted samples (from 2 to 0 ng of IgA) on the gel in triplicate.
Representative full blots including the patterns of OGIgAB represented in pictures 1 and 2 are supplied in Supplementary Data 1.
To assay the limit of blank and the limit of detection, diluted CSF and serum samples with an extremely low quantity of IgA (0 ng and 0.25 ng respectively) were analyzed.

For the first dimension, 17 cm pH 4–7 L IPG strips (Bio-Rad, Hercules, CA, USA) were used. The samples were passively rehydrated overnight using a rehydration buffer containing 7 M urea (Bio-Rad), 2 M thiourea (GE healthcare, Uppsala, Sweden), 2 % CHAPS (GE Healthcare), 1 % IPG buffer (pH 4–7) (GE healthcare), 0.2 % DTT (Bio-Rad) and bromophenol blue (Bio-Rad). The rehydrated IPG strips were focused using PROTEAN IEF cell (Bio-Rad). The current was applied in 3 steps; 250 V was applied in linear ramp for 20 min, then 10,000 V for 2.5 h in linear ramp and finally, the current increased rapidly to 40,000 V/hr when the isoelectric focusing was achieved. The strips were stored at −80 °C until the second dimension gel electrophoresis was performed. Strips were then equilibrated in two steps for 15 min each using an equilibration buffer consisting of 0.1 M Tris HCl (Amresco, Solon, OH, USA) pH 8.8, 6 M urea (Bio-Rad) 30 % Glycerol (GE Healthcare), 4 % SDS (GE Healthcare), 0.002 % bromophenol blue (Bio-Rad). In the first step of equilibration, 26 mM DDT (Bio-Rad) was added to the buffer, while 0.38 M of Iodoacetamide (Bio-Rad) was added to the buffer in the next step. The second dimension was then performed on 10 % SDS–polyacrylamide gels.

Isoproterenol hydrochloride and protease inhibitor cocktail were purchased from Sigma Chemical Co., St. Louis, MO, USA; while the chemicals used for biochemical assays were products of E. Merck Ltd., Mumbai, India. All other chemicals and reagents used were procured from Hi-Media, Mumbai, India. Thio-barbituric acid (TBA), sodium dodecyl sulphate (SDS), Ellman’s reagent and m-phosphoric acid were obtained from E. Merck Ltd., Mumbai, India. While assay kits for CK-MB, LDH, AST and ALT were purchased from Teco Diagnostic,USA; for total cholesterol, Triglyceride and HDL-C, kits were obtained from Span diagnostics Pvt. Ltd., Surat, India. The TNF-α and IL-6 specific ELISA kits were from Ray Biotech, Inc., USA and cardiac troponin-I (cTnI) kit was procured from the Ortho-Clinical Diagnostics, Inc. New York, USA. While Bisacrylamide, Tris hydroxymethyl aminomethane (Tris), glycine, N,N, NU,NU-tetramethylethyldiamide, ammonium persulfate, glycerol, ultra pure urea, 2D cleanup kit and 2D Quant kit were purchased from GE Healthcare, Amersham, Freiburg, Germany; Acrylamide, dithiothreitol, 3–3–1-propane-sulfonate, agarose and Iodoacetamide were purchased from Fluka BioChemika, Buchs, Switzerland.

Acrylamide, bisAcrylamide, DTT, iodoacetamide, CHAPS, SDS, urea, glycerol, thiourea,
TEMED, ammonium persulfate (APS), molecular markers and IPG buffer were obtained from GE
Healthcare Life Sciences (São Paulo, SP, Brazil). Triton X-100, BSA and CBB were obtained
from SIGMA-ALDRICH (São Paulo, SP, Brazil). Trypsin was obtained from Promega (São
Paulo, SP, Brazil).

Acetonitrile (ACN), acetone, trifluoroacetic acid (TFA), trichloroacetic acid (TCA), DL-dithiothreitol (DTT), iodoacetamide (IAA), glycine, EDTA, Tris, endonuclease, phosphoprotease and protease inhibitors, lanthanum chloride, potassium dihydrogen phosphate, Coomassie Blue G-250, imidazole, alkaline phosphatase were purchased from Sigma (Sigma Aldrich St.Louis, MO, USA); urea, CHAPS, SDS, glycerol, acrylamide, ampholine, and Ficoll-Paque™ were purchased from GE healthcare (Uppsala, Sweden); Agarose, Pro-Q® Diamond Phosphoprotein Enrichment kit, Pro-Q® Diamond dye, SYPRO® Ruby and PeppermintStick™ Phosphoprotein Molecular Weight Standards were from Invitrogen™ (Carlsbad, CA); piperazine di-acrylamide (PDA), TEMED, Bio Rad Protein Assay, IPG strips, were from Bio-Rad Laboratories (Hercules, CA). Trypsin (sequencing grade modified) was from Promega (Madison, Wisconsin, USA). All solvents used were Ultra-Resi-Analyzed grade.

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).

Bactopeptone, bis-benzimide, glutaraldehyde, penicillin, propidium iodide, sodium citrate, streptomycin, trypsin, and yeast extract were acquired from Sigma Aldrich (Saint Luis, MO, USA); anti CD61-PerCP, anti CD62-PE, and PAC-1-FITC (Becton-Dickinson, Franklin Lakes, NJ, USA); Hoechst 33342 from Santa Cruz Biotechnology (Dallas, TX, USA); McCoy’s 5A medium and fetal bovine serum (FBS) from Biowest (Nuaillé, France) and XTT test from Biotium (Fremont, CA, USA). Bromophenol blue, dithiothreitol (DTT), glycerol, iodoacetamid, Immobiline Dry Strip gels, pH 4–7, Precast 12.5% polyacrylamide gel and buffer for 2-D DIGE, sodium dodecyl sulfate (SDS), Tris-HCl, UREA, (GE Healthcare, Little Chalfont, UK). Refraction-2D Labeling Kit and DyeAgnostics was obtained from LKB Biotech, Warsaw, Poland. All other reagents were obtained from POCH SA (Gliwice, Poland). Standard polystyrene flasks (T-75 flasks) and flat bottom cell culture microplates (12, 24, 48 and 96 wells) were from TPP Techno Plastic Products AG (Trasadingen, Switzerland). All other disposables were from VWR Int. (Gdansk, Poland).