Immunoblotting of Helicobacter pylori Neutrophil-Activating Protein

Immunoblotting was performed essentially the same as previously described [23 (link)]. SDS-PAGE-separated proteins were transferred onto a polyvinylidene difluoride (PVDF) membrane. The membrane was blocked with 5% nonfat milk in Tris-buffered saline/Tween-20 (TBST) containing 50 mM Tris‐HCl, pH 7.4, 15 mM NaCl, and 0.1% Tween‐20 at room temperature for 1 h. The membrane was then probed with hybridoma culture supernatants containing mouse monoclonal antibody MAb 16F4 against HP-NAP [26 (link)] with a dilution factor of 1:200 in TBST containing 5% bovine serum albumin (BSA) at 4°C overnight. The membrane was washed three times with 5% nonfat milk/TBST, for 10 min each time, and then probed with horseradish peroxidase-conjugated anti-mouse secondary antibody (Jackson ImmunoResearch, West Grove, PA, USA) at a dilution of 1:5000 in 5% nonfat milk/TBST at room temperature for 1 h. After the membrane was washed three times with TBST for 10 min each, the signal of HP-NAP on the membrane was visualized by an enhanced chemiluminescence (ECL) system (PerkinElmer, Waltham, MA, USA) and detected by LAS-3000 imaging system (Fujifilm, Tokyo, Japan).