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S. G. Remington, J. M. Crow, J. D. Nelson; Lipocalin-1 Associates With Lysozyme and With Endogenous Antibodies in Tears. Invest. Ophthalmol. Vis. Sci. 2007;48(13):1907.
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To address the association of lipocalin-1 with other proteins in ocular tears.
1) Western blots: Human tear samples were separated by polyacrylamide gel electrophoresis (PAGE) in 15% gels under reducing and non-reducing conditions, and transferred to nitrocellulose. The blots were sequentially incubated with anti-human lipocalin-1 primary antibodies (R&D Systems), alkaline phosphatase-conjugated secondary antibodies (Sigma), and NBT/BCIP.2) Immuno-selection: Human tears were incubated with anti-human lipocalin-1 antibodies and Protein G sepharose (Amersham Biosciences). The immune complex proteins were eluted, separated by PAGE and silver stained.
1) In Western blots, antibodies to lipocalin-1 recognized reduced lipocalin-1 monomer (17 and 18kD bands), and non-reduced lipocalin-1 dimer (37kD). In heated, non-reduced tear samples, antibodies to lipocalin-1 also recognized other higher molecular weight protein complexes.2) Immuno-selection of lipocalin-1 resulted in co-selection of lysozyme. Controls with no added antibodies also resulted in immuno-selection of lipocalin-1 and lysozyme, implying that normal tears contain antibodies to lipocalin-1 and/or lysozyme.
Lipocalin-1 associates with lysozyme and other proteins in tears. Normal tears contain antibodies to lipocalin-1. Auto-antibodies in tears may be an attribute of normal ocular homeostasis.
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