|Isolation, fractionation and purification of mucins [128,129,130]||- Required steps for all initial molecular/biochemical characterizations.||- Labor intensive and may be expensive.|
- Loss of sample during the process, and therefore cannot be used solely for quantification purposes.
- Care must be taken that the mucins do not become degraded.
|Glycosylation analysis  and mass spectrometry .||- Provides valuable information on species- and organ-specific glycosylation and post-transcriptional modifications of mucins.|
- Precise qualitative and quantitative information on different molecules in the sample, most often proteins and carbohydrates.
|- Expensive system and materials.|
- Identification of proteins/molecules require protein libraries for each animal species of interest and knowledge about glycosylation sites.
|Viscoelasticity of mucus (laser/light scattering analysis , direct rheometry , and fluorescence recovery after photobleaching (FRAP) [13, 133,134,135])||- Laser scattering or quasi-elastic (dynamic) scattering is used specifically for molecular size distribution and for mucin conformation and chain dimensions analysis.|
- FRAP assay is easily applicable for in vitro/ex vivo studies.
- Viscosity/elasticity under shear stress conditions can be done directly in rheometer machines but requires higher amounts of sample.
|- Use mathematical modeling equations to calculate the viscoelasticity, which can introduce errors if not performed or calculated correctly.|
- They do not give information on the quantity or specificity of single mucin component.
- FRAP and other microrheology techniques depend on the diameter and non-adhesiveness of labelled particles used.
|Chromatography separation and detection [128, 129, 136]||- specific technique for separation and molecular analysis of biological substances.|
- Provides information on molecular charge and size.
|- Chromatography is expensive and labor-intensive.|
- If radioactive detection use, handling and disposal, is expensive and environmentally unfriendly.
|Metabolic labeling/ Radiolabel discharge measurement or autoradiography [129, 137].||- Can be used to measure amount of secreted radioactive isotope-labelled substance (e.g. 3H-D-glucoseamine, or iodo[−14C]acetamide) incorporated easily in the newly produced mucins.|
- Historically used for characterization of mucin size in chromatographically separated fractions and for quantitation of total mucus secretion after treatments (as ratio of radioactivity detected at baseline and after treatment).
|- Requires radioactive substance handling and exposure.|
- Materials are strictly regulated and expensive to dispose of.
- Not very sensitive to specific mucin secretion.
- Applicable only in vitro/ex vivo and on sputum samples from patients.