[165] Techniques for the release of nascent protein from ribosomes

Publisher Summary This chapter discusses various techniques used for the release of nascent protein from ribosomes. In the normal course of synthesis, the growing peptide chain is released from the ribosome upon completion of the primary structure to become a soluble protein no longer associated with the biosynthetic apparatus. For the preparation of ribosomes containing labeled nascent protein, unwashed reticulocyte ribosomes resuspended in 0.25 M sucrose may be used for preincubation with a radioactive amino acid to introduce a 14 C label into the ribosomal bound nascent protein. An alternate procedure for the preparation of ribosomes containing 14 C-labeled nascent protein is conducted by using the intact reticulocyte rather than a cell-free preparation, where washed reticulocytes are incubated in a reaction mixture. Both procedures permit a parallel preparation of ribosomes, which have been preincubated with unlabeled amino acids only. The unlabeled ribosomes obtained in this manner may then be incubated with valine- 14 C to determine incorporation of free amino acids into protein under conditions identical to those to be used for the study of release of completed protein from the labeled ribosome.