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A. Keech, E. Joyce, M. Senchyna, L. Jones; A Rapid Method for the Extraction of Mucin from Hydrogel Contact Lenses . Invest. Ophthalmol. Vis. Sci. 2006;47(13):119.
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© ARVO (1962-2015); The Authors (2016-present)
To optimize a technique for the extraction of mucin from hydrogel contact lenses using an in vitro model.
Two sets of Proclear Compatibles (Omafilcon A, Coopervision) contact lenses were incubated for 14 days in an artificial tear solution containing ß–lactoglobulin, human albumin, hen egg lysozyme, bovine lactoferrin, porcine mucin and mixed lipids. Lenses were removed and immediately frozen at –70°C. After thawing, one set of lenses was placed in 2ml of 50:50 Methanol:0.2% Trifluoroacetic acid extraction buffer for 24 hours. The resultant solution was then lyophilized and frozen at –70°C. The other set of frozen lenses was placed on glass plates, where 10µl of Tris/SDS extraction buffer (1% SDS, 50mM Tris, protease inhibitor) was added to the surface. Lenses were then cut into ∼1mm squares and placed in 600µl eppendorf tubes. After an additional 35µl of Tris/SDS extraction buffer was added, the tube was boiled for 10 minutes and then spun at 14,000g for 6 minutes. The supernatant was collected in a separate eppendorf and frozen for future use. Total protein from both extraction methods was quantified using the MicroBCA protein assay. Western blotting of total mucins was performed to ensure extraction of mucin from contact lens.
The total protein yield for in vitro doped lenses subjected to Tris/SDS extraction buffer ranged from 5µg to 24µg. The 50:50 methanol:0.2% Trifluoracetic acid extraction buffer yielded 0.75µg to 12µg of total protein. In addition, qualitative results from western blotting also indicate a much higher yield of total mucins using the Tris/SDS extraction buffer. Exploratory work undertaken on ex vivo lenses confirms that total mucin can be extracted from worn lenses.
We conclude that combining the use of Tris/SDS extraction buffer with the processing techniques developed by our laboratory results in a quick and efficient method for the removal of mucins from hydrogel contact lenses. This method should prove useful in assessing the deposition of mucin on hydrogel contact lenses.
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