Simple and accessible methods for quantifying isolated mucins for further evaluation.

In this study, we present a detailed workflow for the isolation, quantitation, and evaluation of mucin proteins. These methods are applicable to a variety of biological, mucin-containing samples from the airways and other mucosal organ systems. While this report focuses on the salivary MUC5B protein from the respiratory system, the presented methodologies can be applied to other mucins, contributing to a broader application of these techniques. We used a simplified isopycnic centrifugation to purify and enrich MUC5B from human saliva. Isolated MUC5B was then subjected to a Bradford protein assay using a bovine submaxillary mucin (BSM) standard, which more accurately reflects the mucin concentration in our samples compared to a bovine serum albumin (BSA) standard. Additionally, we compare the mucin levels following quantitation using agarose polyacrylamide gel electrophoresis. Our findings show a near 2-fold increase in quantitation from the more representative, BSM standard, suggesting its importance for mucin studies. These methods support a wide range of experimental applications looking to assess mucins, thereby contributing to the broader field of mucin studies and advancing our understanding of the implications of mucins in health and disease.•A streamlined, one-step isopycnic ultracentrifugation to isolate MUC5B from human saliva•A Mucin Bradford assay that is modified from existing Bradford assay techniques to better quantitate mucin for mucin studies•An agarose-polyacrylamide gel electrophoresis method used to visualize and confirm the isolation and quantitation of mucin.