Table 4 Advantages and limitations of quantitative and semi-quantitative protein detection methods for mucus/mucin measurement
Method | Advantages | Limitations |
|---|---|---|
- Used for quantitative determination of mucus viscosity and water/solids ratio by measurement of the decrease in weight of mucus samples after oven drying. - Simple and inexpensive. | - Not an exact measurement of mucin, as the percent dry matter may increase/decrease due to changes in non-mucin molecules (e.g. inflammatory-cell derived products). | |
- Simple and relatively sensitive detection/quantitation of proteins in liquid samples. - Can be used for in vivo collected sputum and ASL. | - Antibody needs to be specific for mucin of interest and epitope should avoid homologous regions/repeats between mucins. - A purified species-specific mucin standard should be used, which is not always available. - | |
- Inexpensive and relatively accurate measurement of specific proteins in liquid samples and tissue homogenates. - Can be used together with housekeeping molecules for proper quantitation. - Allows for the detection of normal and modified forms of the same protein (after stripping of initial labeling) | - Antibody needs to be specific for mucin of interest and epitope should avoid homologous regions/repeats between mucins. - Requires denaturation of mucins for running on SDS-PAGE gels or agarose gels for proper separation of the larger molecules. | |
Dot-blot (Slot-blot) assays [120] | - Inexpensive and quick alternative to western blots for antibody comparison and assessment in a large number of samples. | - Does not separate proteins by size. - Not as sensitive as western blot (quantification is based on intensity image analysis of dots). - Does not typically utilize housekeeping proteins to normalize the signal intensity. |