Estimation of separation of OS and IS membranes using TOM20, Na⁺/K⁺ ATPase α subunit and calnexin as marker proteins.

<p>(A) Specificity of antibodies used to detect TOM20, Na⁺/K⁺ ATPase α subunit and that of anti-calnexin antiserum. Purified rod membranes containing 200 pmol of rhodopsin and cone membranes containing 6 pmol of cone total pigments were subjected to SDS-PAGE and were stained with Coomassie Brilliant Blue (left panel) or probed with antibodies or antiserum against each protein (right three panels). (B-D) Quantitative immunoblot analyses of TOM20 (B), Na⁺/K⁺ ATPase α subunit (C) and calnexin (D). In the upper panels in each of (B)—(D), purified rod membranes containing 200 pmol of rhodopsin or purified cone membranes containing 6 pmol of total cone pigments (Initial), upper and lower membrane fractions obtained from the same number of the purified cells (Upper and Lower, respectively), and a diluted series of initial rod and cone membranes were subjected to SDS-PAGE. These membranes were probed with antibodies or antiserum against each marker protein. To detect the amounts of target proteins precisely, 4 times volume of samples were applied when necessary (4×). The amount of a target protein in each of the membranes was determined with a calibration line obtained from immunoblot signals obtained in a diluted series of initial rod or cone membranes. In the lower panels in each of (B)—(D), examples of quantification are shown. The quantity of a target protein in each fraction is indicated by an arrow in lower panels. With this estimation, one can determine how much % of the target protein is present in each of the membranes as compared with the amount in the initial rod or cone membranes of the same cell number.