title: A pull-down method with a biotinylated bait protein prepared by cell-free translation using a puromycin linker.
abstract: In this paper, we demonstrate a novel pull-down method that dramatically reduces the cost and preparation time of a bait protein by cell-free translation with a puromycin linker. With the C-terminus of the bait protein linked to biotin through a puromycin molecule after the translation reaction and subsequent mRNA degradation by RNase, the prey protein was easily pulled down by streptavidin-coated magnetic beads in a test tube. Three fluorescent prey protein types were tested and confirmed by gel electrophoresis to be pulled down easily and rapidly, depending on their affinity.
authors.affiliation: Graduate School of Science and Engineering, Saitama University, Sakura-ku, Saitama 338-8570, Japan.
authors.forename: Yuki
authors.initials: Y
authors.lastname: Mochizuki
authors.forename: Fumiaki
authors.initials: F
authors.lastname: Kohno
authors.forename: Koichi
authors.initials: K
authors.lastname: Nishigaki
authors.forename: Naoto
authors.initials: N
authors.lastname: Nemoto
mesh_terms: D001710:Biotin / Q000737:chemistry / Q000378:metabolism*; D002474:Cell-Free System; D019793:Fluorescein / Q000737:chemistry; D008280:Magnetics; D011485:Protein Binding; D014176:Protein Biosynthesis; D025941:Protein Interaction Mapping; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*; D011691:Puromycin / Q000737:chemistry*; D012333:RNA, Messenger / Q000378:metabolism; D019809:Streptavidin / Q000737:chemistry
chemical_list: D011506:Proteins; D012333:RNA, Messenger; D011691:Puromycin; D001710:Biotin; D019809:Streptavidin; D019793:Fluorescein