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This website isÂ .MS2 tag-mediated immunoprecipitation and affinity purification of protein complexes.
Ams2 can bind to its cognate target sequence in a very efficient manner as the result of an efficient ternary complex formation involving a two-step recognition event. In this chapter, we describe the usage of a tag peptide fused to the am2 gene (Ams2-N) to allow isolation of target proteins specifically interacting with the cognate target sequence of Ams2. In the first step, in vitro translation of the mRNA fused to the am2-N peptide results in the formation of a ternary complex composed of the mRNA, the am2-N peptide, and Ams2 protein. In the second step, both am2-N and target proteins can be isolated by precipitation and affinity purification through use of the am2-N peptide, respectively. We also describe a procedure that allows efficient isolation of target proteins which are covalently crosslinked to their cognate target sequence by an internal am2-N-tagged RNA strand, which was formed in vitro from an am2-N-tagged template RNA by a bacterial RNase III. This procedure allows the rapid isolation of target proteins with a single base-pair (R1 RNA-R1 RNA) or a loop (R1 RNA-R1 DNA) mismatch in their target sequence, which will be useful for elucidating the mismatch repair mechanism as well as the RNA-dependent RNA polymerase activities.Here is what happened. After a Mother’s Day garden party, a partygoer went into the women’s restroom. She then followed a young woman into the stall where another young woman was sitting. The partygoer closed the door and locked it. After several minutes the partygoer began to undress and talk to the young woman. The young woman was not undressed for very long before the partygoer began to undress, reaching under the young women’s shorts and bra. The partygoer began to fondle the young woman’s breasts and she in turn began to fondle the partygoer. The young woman eventually orally copulated the partygoer, moving down to her vagina and performing oral sex