Plasma Protein 2DE Western Blot

Plasma proteins (0.5 mg) were run by 2DE (cm 2DE, using 13-cm strip pH 4–7). Proteins were transferred (2 h, 28 V) from the gel onto PVDF membrane (Hybond P, 0.2 μm) using two sheets of thick paper (Bio-Rad, Hercules, CA, USA), saturated with 48 mM Tris, 39 mM glycine, 0.037% SDS, 20% ethanol. The membrane was treated following a protocol of Blue Dry Western [36 (link)] and treated with antibodies [21 (link)]. Primary antibodies were mouse monoclonal anti-Hp (C8, sc-376893, or F8, sc-390962, from “Santa Cruz Biotechnology”, Santa Cruz, CA, USA) in dilution 1/25 (80 ng/mL in TBS (25 mM), Tris (pH 7.5) and 150 mM NaCl containing 3% (w/v) BSA) or rabbit polyclonal anti-Hp (MBS177476, MyBioSource, San Diego, CA, USA). Secondary goat anti-mouse immunoglobulins G labeled by horseradish peroxidase (NA931V, “GE Healthcare”, Chicago, IL, USA ) were used in TBS containing 3% (w/v) nonfat dry milk (1/5000 dilution). The reaction was developed using ECL (Western Lightning Ultra, “PerkinElmer”, Waltham, MA, USA) and X-ray film (Amersham Hyper film ECL).