With regards to mucus production, do you have any procedures to examine the mucus and periciliary fluid proteins in ALI cultures that lend to western analysis. E.g. collection and processing for protein. What would you suggest would be a good reference gene for loading control (MUC5?) in western analysis.

Answer

If you are interested in the secreted mucin, you can add warm PBS to the apical chamber,  incubate for 20 minutes and then collect the PBS with mucin. Acetone can be used to precipitate the protein, followed by standard western blotting procedure. You can also collect the whole culture using protein lysis buffer followed by standard western blotting  procedure. For the secreted mucin, Muc5AC and Muc5B are good markers to use. For the membrane-associated mucin, Muc1 is a good marker to use.