Electrophoresis and western blot of BAL fluid were performed as previously described using precast sodium dodecyl sulfate (SDS) acrylamide gels (4–15% gradient) from BioRad (Hercules, CA), under reducing or non-reducing conditions as noted, and protein transfer to nitrocellulose membrane.24 (link) Nitrocellulose membranes were incubated for 2 h with mAbs or polyclonal antisera specific for YM-1, also known as mouse Chitinase 3-like 3, from R&D Systems (Minneapolis, MN), CLCA3, also known as mouse CLCA1, from Santa Cruz Biotechnology, Inc. (Dallas, TX), biotin anti-mouse IL-12/IL-23 p40 from BioLegend (San Diego, CA), polyclonal HRP-anti-IgG Fc (Santa Cruz Biotechnology, Inc.), or biotinylated anti-MDI DA5 developed in our laboratory.25 (link) After extensive washing with PBS containing 0.05% Tween 20, strips were incubated with appropriate secondary antibody and developed with enhanced chemiluminescence substrate from Thermo Fisher Scientific Inc. (Rockford, IL).
Protocol detail
Western Blot Analysis of BAL Fluid
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Acrylamide
Anti igg
Antibody
Antisera
Biotin
Chemiluminescence
Chitinase
Electrophoresis
Gels
Il 12
Mabs
Membranes
Mouse
Nitrocellulose
Protein
Sodium dodecyl sulfate
Tween 20
Western blot
Corresponding Organization :
Other organizations : Yale University
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Variable analysis
independent variables
- Reducing or non-reducing conditions for the SDS-PAGE and western blot
- Presence and levels of the proteins YM-1 (mouse Chitinase 3-like 3), CLCA3 (mouse CLCA1), and IL-12/IL-23 p40 in the BAL fluid samples
- Precast sodium dodecyl sulfate (SDS) acrylamide gels (4–15% gradient) from BioRad used for electrophoresis
- Nitrocellulose membrane used for protein transfer
- Incubation time (2 hours) for the nitrocellulose membranes with the specific antibodies
- Washing of the strips with PBS containing 0.05% Tween 20
- Use of appropriate secondary antibodies and enhanced chemiluminescence substrate for detection
- Polyclonal antisera specific for YM-1, CLCA3, and biotinylated anti-MDI DA5 developed in the authors' laboratory
- Biotin anti-mouse IL-12/IL-23 p40 from BioLegend
- Not explicitly mentioned
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