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Table 4 Advantages and limitations of quantitative and semi-quantitative protein detection methods for mucus/mucin measurement

From: Strategies for measuring airway mucus and mucins

MethodAdvantagesLimitations
Percent solid matter [13, 88]- 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).
ELISA [117, 118]- 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.
-
SDS-PAGE/western blot assay [100, 119]- 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.