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. 1997 Dec;8(12):2365–2378. doi: 10.1091/mbc.8.12.2365

Figure 1.

Figure 1

Specificity of antibody preparations. (A) Preparations of purified recombinant G protein α subunits (25 ng) were resolved by SDS-PAGE and analyzed by silver staining or Western immunoblotting. Only the region of the gel where G protein α subunits migrate is shown (7 cm long, 11% polyacrylamide gel). If the film is exposed to the blots for an extended time (not shown), a reaction with αs can be observed with the A569 antibody preparation (but not with B087 or R4). (B) Crude membrane fractions (25 μg protein) were resolved on a 16-cm long, 9% polyacrylamide gel, and immunoblots are shown. Numbers at left indicate the position of prestained molecular mass standards in kilodaltons. The crude membrane preparations are from MDCK (vector control cells, lanes 1), MA104 (lanes 2), or fibroblasts (lanes 3). The α subunit reactivities of the antibodies are indicated in parentheses near the top of the panel. A569 detects only αi in cell membranes because it reacts better with αi than αs and there is approximately 10-fold more αi than αs in most cells (and there is little or no αo expressed in these cells). Mab R4 is specific for αi1 (X. Li et al., 1995). Monoclonal antibody R4 does not react well for immunoblotting and is not sensitive enough, by this method, to detect αi1 in MDCK cells or fibroblasts; only MA104 membranes are shown for this antibody. The polyclonal caveolin antibody preparation reacts primarily with one protein band but also with other minor isoforms of caveolin in the 21–25 kDa region of the blot. Please note that the 40-kDa band labeled αi (to the right of lane 3 of the caveolin blot, panel B) is residual signal remaining from a previous incubation of the blot with the αi reactive A569 antibody preparation and is not from reactivity with the caveolin antibodies. Antibodies for Western immunoblotting were as follows: affinity-purified A569 and B087 at 100 ng/ml, the purified polyclonal caveolin antibodies at 500 ng/ml, culture medium from monoclonal antibody R4-producing cells diluted 1:25 (vol/vol).