FIGURE 1.

Purification of TAP. A, purification of TAP with different detergents. TAP-containing membranes (5 mg/ml of protein) were solubilized by different detergents and purified by metal affinity chromatography. Aliquots of membranes (M), solubilization (S), flow through (FT), wash with 20 mm (W1), wash with 40 mm histidine (W2), and elution with 200 mm of histidine (E) were analyzed by SDS-PAGE (9%, Coomassie-stained) and immunoblotting using monoclonal α-TAP1 (148.3) and α-TAP2 (435.3) antibodies. B, stoichiometry of TAP. TAP-containing membranes (10 mg/ml protein) were solubilized by 2% digitonin, purified by the His₁₀ tag at the C terminus of TAP1 via metal affinity chromatography, and subsequently immunoprecipitated via monoclonal α-TAP2 (435.3) antibody to isolate exclusively heterodimeric TAP complexes or α-MHC I (HC10) antibody as isotope control. Aliquots of membranes (M), solubilization (S), metal affinity-purified TAP (P), and immunoprecipitated TAP (IP) were analyzed by SDS-PAGE (10%) and immunoblotting using monoclonal α-TAP1 (148.3) and α-TAP2 (435.3) antibodies. The ratio of the TAP1 and TAP2 intensities in each fraction was normalized to the membrane level. The ratios depict the mean of three independent experiments.