Autoradiogram showing the 12-kD protein that is cross-linked by an ¹²⁵I-labeled arylazido inositolphosphate analogue. The 110 fractions from the Sephacryl S-300 gel filtration column were divided into 11 groups of 10 each and combined and concentrated (6-fold). These preparations were then loaded onto 3% to 17% gradient, nondenaturing (Nonidet P-40) polyacrylamide gel. After development, gel segments containing slowly migrating aggregates or complexes (estimated at 400 to 600 kD) were cut from the gel and eluted. After dialyzing each eluate against 50 mmol/L HEPES buffer (pH 7.5) containing 0.1% Nonidet P-40 to remove sulfhydryl reagents, 125 μL aliquots were preincubated for 60 minutes with the¹²⁵I-labeled probe (2.0 × 10⁷ dpm) and then irradiated (254 nm) for 2 minutes at a distance of 3.5 cm using a Mineralight UVSL source (Ultra-violet Products, San Gabriel, CA). Unreacted azido reagent was destroyed with 5% 2-mercaptoethanol in 50 mmol/L HEPES containing 0.1% Nonidet P-40. Reaction mixtures were then applied to 3% to 17% gradient, nondenaturing (Nonidet P-40) polyacrylamide gels that were used to prepare autoradiograms. Gel segments containing the slowly migrating or the rapidly migrating heavily labeled components demonstrated for fractions no. 41 through 50 were cut from the nondenaturing gel and inserted at the top of a 10% to 20% gradient SDS polyacrylamide gel. Autoradiograms prepared from the SDS gels identified a labeled 12-kD protein derived from the rapidly migrating component (BOTTOM) and a labeled 12-kD protein derived from the slowly migrating component (TOP). Positions of molecular mass markers are indicated on the left.