Fig. 6
Relative in vitro binding of recombinant αAN101D and WTαA proteins to lens membrane. a The recombinant WT αA- and αA-N101D proteins were labeled with Alexa 350, purified by a size-exclusion HPLC column and analyzed by SDS-PAGE. Lane 1: Coomassie blue-stained WTαA, lane 2: αAN101D mutant protein, and lane 3: purified lens membrane from non-transgenic C57 mice. b Images of labeled αAN101D and WTαA proteins. Lane 1: Alexa 350-labeled WT αA, and lane 2: αAN101D protein. c Binding of a WT αA, and αAN101D with purified lens membrane (2.5 mg protein; isolated from 1- to 3-month old non-transgenic C57 mice). During the binding assay, the protein mixtures were incubated with increasing but identical concentrations of either Alexa-labelled WTαA- or αA-N101D at 37οC for 6 h, centrifuged at 14,000Xg and the supernatant and pellet (membrane fraction) recovered. After washing the membrane fraction with water and centrifugation as above, the relative fluorescence of membranes incubated with WT αA- and αA-N101D mutant proteins was determined. The values reported are the average of triplicate assays