SDS-PAGE and immunoblot transfers were performed as described by the manufacturer for 10% Bis-Tris gels using 3-(
N-morpholino)propanesulfonic acid (MOPS) running buffer (Novex; Invitrogen, Carlsbad, CA). To identify the 58-kDa protein species in whole corneal extracts from mouse as MSA, SDS-PAGE (−DTT) was performed, the gel was stained (Gel Code Blue; Pierce, Rockford, IL), and a gel slice was cut that contained the 58-kDa band. Mass spectra analysis of trypsinized fragments of this species was performed as a service (Harvard Microsequencing Center, Cambridge, MA). Digital images were obtained under UV light (ChemiImager 4000; Alpha Innotech Corp., San Leandro, CA) from fluorescence-stained immunoblots or red-stained (SyproRuby or SyproRed; Molecular Probes, Inc., Eugene, OR) gels. One of the red stains (SyproRed; Molecular Probes) was used because it generates a linear signal over a more extensive protein concentration range with less dependence on amino acid composition than Coomassie blue staining.
17 For quantitative analysis, bracketed exposures were taken, and the exposure with the strongest signal that still appeared to be linear with respect to exposure length was used. Band signals from these red-stained gels were quantitated with the imaging software (ChemiImager; Alpha Innotech Corp.) in an average of six experiments. For immunoblots, membranes were blocked (SeaBlock; Pierce) for anti-serum albumin antibodies or 5% dry milk and 0.1% Tween-20 in 1× PBS for anti-TKT
11 and anti-aldehyde dehydrogenase 3a1 (ALDH3) antibodies (the kind gift of Ronald Lindahl, Department of Biochemistry, University of South Dakota, Vermillon, SD). Albumin antibodies included two polyclonal rabbit anti-MSAs (RDI-MSALBabr; Research Diagnostics, Inc., Flanders, NJ, and 55442; ICN Pharmaceuticals, Inc., Costa Mesa, CA), a monoclonal mouse anti-BSA (B2901; Sigma-Aldrich, Inc., St. Louis, MO) and a polyclonal rabbit anti-BSA (56963; ICN Pharmaceuticals, Inc). Secondary antibody incubations with horseradish peroxidase linked to donkey anti-rabbit or anti-mouse Ig (Amersham Pharmacia Biotech, Piscataway, NJ) were diluted 1:3000. Enhanced chemiluminescence (ECL) detection was as described by the manufacturer (Amersham Pharamcia Biotech).