To search for novel proteins that bind to the UBA domain of p62, we performed in vitro expression cloning (IVEC) using the ProteoLink IVEC system from Promega (Cat. No. L6500). The human adult brain library 96 well plates with 100 cDNAs per well were transcribed and translated employing the Gold TNT SP6 Express 96 plate in the presence of [ 35 S] methionine and ubiquitin (25 µg/µl, Sigma). By combining 4 protein pools as one mixed pool, 96 protein pools were divided into 24 mixed pools ( Fig. 2A,  2B ). Each lane contained more than 100 proteins (theoretically 400) with different molecular weight. Therefore, each lane appeared as a smear, indicating that the in vitro transcription/translation system from Promega worked successfully. In order to examine whether proteins synthesized in the IVEC system are also ubiquitinated, Western blot analysis was performed by blotting the newly synthesized proteins (in the presence of cold methionine instead of 35 S methionine) with ubiquitin monoclonal antibody. In the mixed protein pools, each of the 24 lanes appeared as a smear, indicating that proteins synthesized by the IVEC system are also ubiquitinated (Fig. 3A) . Furthermore, the rabbit reticulocyte lysate in the IVEC system can utilize different lysine linkages of ubiquitin (i.e., Ub K29, Ub K48, and Ub K63) for ubiquitination (Fig. 3B) . In order to investigate whether the agarose-immobilised p62 UBA peptide has binding specificity, a mixed protein pool synthesized by IVEC system was tested in a pull down assay in the presence of agarose beads alone or in the presence of p62 UBA agarose beads (Fig. 3C ). Our results revealed that proteins that bound to p62's UBA domain could not be pulled down by agarose beads alone, indicating that the agarose-immobilised p62 UBA peptide had binding specificity. 