Monoclonal Anti-MUC5AC antibody produced in mouse

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	-Mouse (1:1000)

Protocol tips
-Mouse (1:1000)

Publication protocol

For total protein extracts preparation, cells (about 2 × 106) were lysed in 100 μL of Laemmli sample buffer [1.5% (w/v) SDS; 5% (v/v) glycerol; 0.001% (w/v) bromophenol blue; 0.5 mM dithiothreitol (DTT); 31.25 mM Tris, pH 6.8] supplemented with 5 U of Benzonase (Sigma-Aldrich) and 10 mM MgCl2 for nucleic acids degradation. Protein quantification was carried out with the RC DCTM Protein Assay (BioRad, Laboratories, Hercules, CA, United States) according with the manufacturer’s instructions. For mucin detection, 100 μg of total protein extract were vacuum-fixed onto nitrocellulose filters (Schleicher and Schuell, Dassel, Germany) using a Bio-DotTM apparatus (Bio-Rad). For zymogen detection, 100 μg of total protein extract were separated by SDS-PAGE on 12.5% (w/v) polyacrylamide mini-gels and transferred onto nitrocellulose filters. After 15 min wash with 0.1% (v/v) Tween-20 in PBS (PBS-T), membranes were blocked for 2 h in 5% (w/v) skim milk in PBS-T at RT and, then, probed over-night, at 4 °C, with mucin/zymogen specific Ab diluted in the same skim milk solution. Used antibodies: anti-MUC5AC (clone 2H7) mAb (1:1000 diluted); anti-MUC6 (clone H5) mAb (1:5 diluted); anti-PGA5 (clone 4G9) mAb (1:800 diluted); and anti-HGL (H-70) polyclonal Ab (pAb) (1:800 diluted). For immunodetection, blots were incubated for 2 h at RT with the respective secondary Ab (anti-mouse IgG conjugated with HRP (BioRad) with proper dilution for mAb recognition, or anti-rabbit IgG conjugated with HRP (BioRad), with proper dilution for pAb recognition). Finally, blots were developed using the SuperSignal® West Pico Chemiluminescent Substrate detection system (Pierce, Rockford, IL, United States) and exposed to X-ray films (Fuji Super RX 100NIF, Fujifilm, Tokyo, Japan). Four washing steps with PBS-T were always performed between consecutive incubations.

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