Purpose: To identify, separate and characterize the different mucin species in the human pre-corneal tear film. Methods: Tears were collected from healthy non contact lens wearers. Proteins in tears were separated by one of the following methods: 3-15% SDS-PAGE, 1% agarose gel electrophoresis or size exclusion chromatography. Identification of proteins was performed by the following staining methods: silver for total protein, Stains-all® for heavily glycosylated species and by digoxigenin labelling for carbohydrate moieties. Immuno-reactivity of both the protein and glycan components of mucins were identified by Western blotting. Results: SDS-PAGE and size exclusion chromatography has allowed the partial purification and characterization of mucins from human tears. Agarose electrophoresis separated mucins into species of molecular weight in excess of 200kDa and up to approximately 500 kDa and in excess of 700kDa. Differences in species were detected using mAbs to different mucin gene products. The larger mucin species (≥500kDa) were reactive to antibodies to MUC1. Identification and classification of all the other mucin species is currently underway. Sugar analysis of these large molecular weight molecules showed that there were differences in carbohydrate composition of the mucin species. Conclusions: Agarose electrophoresis has allowed the separation of different mucins in the human tear film. The largest mucin species has been positively identified as MUC1.
|Journal||Investigative Ophthalmology and Visual Science|
|Publication status||Published - 15 Feb 1996|