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L.N. Subbaraman, M. Senchyna, L.W. Jones; Stabilization of Lysozyme Mass Extracted from Silicone–Hydrogel Contact Lens Materials . Invest. Ophthalmol. Vis. Sci. 2004;45(13):1556.
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Purpose:Preliminary results in our laboratory demonstrated that lysozyme deposits extracted from silicone–hydrogel (SH) contact lens materials demonstrated a loss in total mass as a function of storage time when assessed by Western blotting (WB). This loss represents a potential source of error when quantifying total lysozyme deposition. Thus, the purpose of this work was to devise a method whereby lysozyme mass would be preserved over time and would be compatible with our previously described WB procedure1. Methods: Lysozyme deposits from human worn lenses were extracted using a 50:50 mixture of 0.2% trifluoroacetic acid and acetonitrile. Extracts were lyophilized to dryness, then resuspended in either Reconstitution Buffer (RB) (10mM Tris–HCl, 1mM EDTA) or Modified Reconstitution Buffer (MRB) (RB + 0.9% saline). BioStab (1 in 4 parts) (Sigma) was added to one half of the samples from each buffer group. 1µL of each of the samples was immediately subjected to SDS PAGE and WB on a PhastSystemTM (Pharmacia), while the remaining volume was aliquoted and stored at –20°C or –70°C and subjected to the same procedures after 48 hours of storage. All WBs were imaged and quantified on a Storm 840TM Imaging system. Comparison of lysozyme band intensity in stored versus fresh samples enabled calculation of percentage mass loss of lysozyme. Results: As can be seen in Table 1, resuspension of lyophilized SH lens extracts in MRB and BioStab provided essentially 100% protection to lysozyme mass when stored in frozen aliquots. Conclusions:We have optimized a procedure using MRB, BioStab and storage at –70oC, whereby the extracted mass of lysozyme deposits found on SH lenses can be preserved without loss to facilitate accurate quantitation via our WB procedure. Table 1: % Mass Loss of Lysozyme After 48 Hours of Storage:
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