Reducing SDS-PAGE (7.5%) analysis of the TCblR protein during various stages of purification. (A) Protein eluted with EDTA from the TC-Cbl-Sephacryl affinity matrix. The 58-kDa region corresponding to the size of TCblR is marked with an arrow. (B) Protein eluted with 0.5 M of MgCl2 from the TC-Cbl affinity matrix. Lanes 1, 2, and 3 represent 3 batches of purification showing similar profiles by silver staining. (C) Elution from the affinity matrix after the purified protein from the first matrix was reapplied to the same matrix. Lane 1, protein retained on the matrix and was eluted with 0.5 M of MgCl2; lane 2, protein in the effluent that was not retained on the matrix. (D) The protein product after the final purification. The receptor-TC-Cbl complex released by photolysis of the Cbl-matrix was applied to a WGA agarose matrix, and the TCblR-TC complex bound was eluted. The upper band corresponds to the expected size of the receptor and the lower band represents TC that was bound to the receptor.
