Western Blot Analysis of MUC5AC Secretion

Human bronchial epithelial cells were lysed in lysis buffer and subjected to SDS-PAGE and a Western blot analysis^{38 (link)}. Blots were reacted for 2 h with monoclonal antibody diluted at 1:1000 and with the respective HRP-conjugated secondary antibodies diluted at 1:2000. After each antibody reaction, membranes were washed 3 times with 0.1% TBS-Tween, and blots were visualized with Super Signal West Pico chemiluminescence substrate (Thermo Fisher Scientific, Inc.). Images were captured by LAS-4000 (GE HealthCare Dharmacon, Inc.). The band intensity was quantified using the Multi Gauge software program (FUJIFILM, Inc., Tokyo, Japan). For the detection of MUC5AC protein secretion into the medium, slot blotting was performed as previously described^{39 (link)}.

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Oniki K., Nohara H., Nakashima R., Obata Y., Muto N., Sakamoto Y., Ueno-Shuto K., Imafuku T., Ishima Y., Watanabe H., Maruyama T., Otake K., Ogata Y., Suico M.A., Kai H., Shuto T, & Saruwatari J. (2020). The DsbA-L gene is associated with respiratory function of the elderly via its adiponectin multimeric or antioxidant properties. Scientific Reports, 10, 5973.

Publication 2020

Super Signal West Pico chemiluminescence substrate Multi Gauge software program LAS-4000

Antibodies Antibody Bronchial Buffer Chemiluminescence Epithelial cells Human Membranes Monoclonal antibody Muc5ac Protein Sds page Secretion Tween Western blot

Corresponding Organization :

Other organizations : Kumamoto University, Sojo University, Tokushima University, Japanese Red Cross Kumamoto Hospital

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Variable analysis

independent variables

Lysis buffer used to lyse human bronchial epithelial cells

dependent variables

Expression/abundance of MUC5AC protein detected by Western blot analysis
MUC5AC protein secretion into the medium detected by slot blotting

control variables

Antibody dilutions (1:1000 for monoclonal antibody, 1:2000 for HRP-conjugated secondary antibodies)
Washing steps (3 times with 0.1% TBS-Tween after each antibody reaction)
Visualization method (Super Signal West Pico chemiluminescence substrate)
Image capture (LAS-4000 imager)
Quantification method (Multi Gauge software)

positive controls

None mentioned

negative controls

None mentioned

Annotations

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