A. WGA lectin affinity chromatography of MDCK cell membrane proteins. Bound proteins were eluted with 0.3 M N-acetylglucosamine, and stained by Coomassie blue after gel electrophoresis. L: aliquot of loaded protein preparation. E: eluted protein pattern (bracket indicates 114 kDa region). B. Flow chart for the purification of gp114. MDCK cell membranes were recovered by high-speed centrifugation from a postmitochondrial supernatant and partially solubilized by treatment with the non-ionic detergent Triton X-100 on ice. Soluble proteins were applied to immunoaffinity columns, and the eluted fractions concentrated by methanol-chloroform extraction-precipitation. Gp114 did not accumulate at the "protein" interface between aqueous and lipid phase but stayed in the hydrophilic supernatant. C. Enrichment of gp114. Lane 1 (A) corresponds to the methanolic phase after chloroform-methanol extraction of eluted proteins from the gp114 immunoaffinity column. Gp114 (arrowhead) is only weakly stained by Coomassie solution. Lane 2 (Ip) contains deglycosylated gp114 (arrowhead) after immunoprecipitation which was confirmed by Western blotting (not shown). The heavy chain of gp114 IgG is indicated by a white arrowhead.