Figure 5

Purification of a native MP20/galectin-3 complex from lens membranes and role of lectin site. (A) SDS-PAGE and Western blot documentation of the purification process. Lane 1: Molecular weight markers. Lane 2: Partially stripped lens fiber cell membranes showing both galectin-3 and enriched MP20. Lanes 3 and 4: Proteins that did not bind (including MP20 and galectin-3) and proteins that did bind to the MonoQ column, respectively. Lane 5: MP20 and galectin-3 eluted from the S-200 column. Lanes 6 and 7: Immunoblot of lane 5 identifying MP20 and galectin-3, respectively. Note that anti MP20 also recognizes dimeric MP20 that is not normally detected on stained gels. (B) Galectin-3 and MP20 did not bind to the column at pH8 (arrow). (C) Size exclusion chromatography of the complex. A single peak eluted from the S-200 column at 13.8 ml. AU refers to absorbance units at 280 nm. (D) Calibration of the S-200 m column. Vo = 8 ml. MP20/galectin-3 complex has an apparent molecular mass of 102 kDa (arrow). (E) Immunoprecipitation of MP20 and galectin-3 and protein detection by Western blotting. Lanes 1 and 2: galectin-3 detected without and with lactose present, respectively. Note the removal of galectin-3 from the complex in the presence of lactose. Lanes 3 and 4: Equal amounts of MP20 are detected without and with lactose present, respectively.