Journal: Methods in molecular biology (Clifton, N.J.)

Article Title: Modulation of Lung Epithelial Cell Function Using Conditional and Inducible Transgenic Approaches

doi: 10.1007/978-1-4939-8570-8_14

Figure Lengend Snippet: Evaluating the effects of transgene expression and gene disruption at the protein level. (a) A dot-blot ELISA was performed on mouse lung lavage fluid applied to a PVDF membrane using a 96-well vacuum manifold (Whatman Minifold I, GE Healthcare Bio-Sciences). A standard curve was generated by applying lavage fluid from a mouse with lipopolysaccharide-induced lung inflammation. Serial twofold dilutions (1:1 vol/vol in 3 M urea/100 mM DTT) were applied to a PVDF membrane. Membranes were blotted with rabbit-anti-Muc5b antisera (1:2000 dilution). Signal detection was performed using an Odyssey CLx Imager (LI-COR Biotechnology); anti-Muc5b-labeled samples were detected using IRDye 680RD goat anti-rabbit IgG, (1:5000 diluted in TBS-formulated LI-COR Odyssey Blocking Buffer). Accurate amounts of Muc5b are not known, but relative levels can be precisely estimated and assigned arbitrary units (a.u.). In the example shown, Scgb1a1-Muc5b transgenic (Tg) and wild-type (WT) littermate controls were compared on dot-blot membranes. (b) Enumerated results of Muc5b dot-blot ELISA are shown in a bar graph plot. Differences were determined by Mann-Whitney U-test comparison of a.u. Muc5b signal intensities. (c) Western blot analysis was used to examine mouse lung lavage samples from Muc5b conditional knockout mice (Muc5bΔ/Δ), which were compared to SFTPC-Cre negative littermate (Muc5blox/lox) and Muc5ac knockout controls. Neat lung lavage samples (25 μL) were diluted in non-reducing 6× loading buffer (5 μL) and separated by electrophoresis in an SDS-agarose gel (42 V, 4 °C, overnight). Samples were transferred to a PVDF membrane, blotted with rabbit-anti-Muc5b antisera (1:2000 dilution) and detected by chemiluminescence using biotinylated goat-anti-rabbit IgG and streptavidin-horseradish peroxidase. Blots were imaged using a ChemiDoc Imaging System (Bio-Rad)

Article Snippet: Please see figure legends for details relating to each protocol ( see Notes 28 – 30 ). fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window caption a7 Evaluating the effects of transgene expression and gene disruption at the protein level. ( a ) A dot-blot ELISA was performed on mouse lung lavage fluid applied to a PVDF membrane using a 96-well vacuum manifold (Whatman Minifold I, GE Healthcare Bio-Sciences).

Techniques: Expressing, Dot Blot, Enzyme-linked Immunosorbent Assay, Generated, Labeling, Blocking Assay, Transgenic Assay, MANN-WHITNEY, Western Blot, Knock-Out, Electrophoresis, Agarose Gel Electrophoresis, Imaging