Figure 11

Procedure to study protein-nucleic acid interactions. The oligonucleotide is designed to contain a 5'-biotin tag, a 3'-fluorescent tag and a UV sensitive group in the middle. (A) Crosslink initiated by exposing to UV (305+16 nm). (B) After three hours of UV activation, denatured samples were subjected to SDS-PAGE analysis, where crosslinked species were confirmed by fluorescent imaging and all bands were visualized by Commassie staining technique. (C) Interesting spots were picked for protease enzymatic in-gel digestion to yield peptide mixtures. (D) Crosslinked peptides were extracted by magnetic streptavidin beads and uncrosslinked peptides were washed away. (E) The crosslinked peptides were subjected to DNase I degradation to minimize the attaching oligonucleotide moieties. (F) Crosslinked peptides with the remaining nucleic acid attached were extracted by reverse phase ZipTip C18 cartridge and analyzed by Q-tof ESI. (G) Raw data were collected and processed by Protein-Lynx to generate a PKL file, which was used as input to CLPM to identify matches with theoretical peptides.