Figure 2

Purification and characterization of lens MP20. (A) SDS-PAGE and Western blot documentation of the purification process. Lane 1: Molecular weight markers. Lane 2: Fully stripped membranes showing significant enrichment in MP20 (20 kDa), and other integral membrane proteins including MIP (26 kDa) and its 22 kDa cleavage product, and MP38 which is the cleavage form of connexins 46 and 50. Lane 3 and 4: Insoluble and soluble fractions, respectively, following treatment of membranes with DM. Lane 5 and 6: Soluble proteins that did not bind (mostly MP20) and proteins that did bind to the MonoQ, respectively. Lane 7: Pure MP20 eluted from size exclusion chromatography column S-200. Lane 8: Immunoblot of lane 5 identifying MP20 and its dimer that is not detected in the stained gel. (B) Single step purification of MP20 on a MonoQ column. MP20 is the only major integral membrane protein that does not bind to the column at pH 8 (arrow). (C) Size exclusion chromatography of pure MP20. The protein was eluted at 15 ml. AU and mAU are absorbance units at 280 nm. (D) Calibration of the S-200 column. Vo = 8 ml. MP20 has an apparent molecular mass of 60 kDa (arrow).