Figure 1.

The organelle trap assay for novel nucleoporins. In the ligand-binding/organelle trap assay, Xenopus egg cytosolic proteins (polygons) are applied to a ligand affinity matrix in the first selection step. In this study, immobilized WGA was used as the matrix. The column is washed, and bound polypeptides (open polygons) are then labeled with biotin (closed circles). The biotin-tagged proteins are eluted from the column with competing ligand, e.g., GlcNAc/TCT buffer in the case of WGA. In the second selection step, the mixture is added to an AL assembly reaction. Only nucleoporins and pore-associated polypeptides (tagged open triangles) will assemble into AL and are identified by Western blotting with SA–HRP. Nonspecific biotin-tagged proteins (tagged open hexagons and squares) are not incorporated and are removed from the AL during centrifugation.