Magicmark xp protein standard

Electrophoresis was performed in a gradient of 4–20% SDS-PAG using Tris-Glycine buffer (Novex, Life Technologies, Carlsbad, CA, USA). Native PAGE was performed in 4–20% gels using Tris-Glycine native running buffer and SDS-free gel loading buffer (Novex, Life Technologies, Carlsbad, CA, USA). Gels were calibrated using PageRuler Plus pre-stained protein ladder and MagicMark XP Protein Standard ( ThermoFisher Scientific, Waltham, MA, USA) or Western blot standard (Serva, Heidelberg, Germany). Proteins were transferred onto 0.2 µm membrane (Protran BA83, Whatman GmbH, Dassel, Germany) at 45 V for 2 h. Membranes were blocked with 6% skimmed milk in PBS with 0.1% Tween 20 (blocking buffer) for 2 h at room temperature or overnight at 4 °C. Incubation with mAbs diluted in blocking buffer (1 µg/mL) was performed for 2 h at room temperature. After five times washing (5 min each) in PBS with 0.1% Tween 20 (PBS-Tween) membranes were incubated for 1 h 30 min with anti-human IgG-HRP conjugate (Dako Laboratories, Glostrup, Denmark) diluted 1:10 K in blocking buffer. The membranes were washed with PBS-Tween as indicated above, treated with Pierce ECL Western blotting substrate (Pierce, Rockford, Tempe, AZ, USA) for 1 min, and exposed to Amersham Hyperfilm ECL (GE Healthcare Limited, Pollards Wood, UK).

Unfiltered and filtered CSF samples were run on a NuPAGE™ 4–12% bis-tris (5 and 100 kDa filtrates) or NuPAGE™ 3–8% tris-acetate protein gel (300 and 750 kDa filtrates) (Thermo Fisher Scientific). MagicMark™ XP protein standard was run on the 4–12% gel and HiMark™ protein standard was used for the 3–8% gel (Thermo Fisher Scientific). Gels were placed in 25% isopropanol with 10% acetic acid for 30 min and then stained in 0.006% Coomassie brilliant blue in 10% acetic acid overnight at room temperature. Following destaining with 10% acetic acid, gels were rinsed in H₂O. Images of gels were captured using Carestream MI system and software.