Notes: This article describes the use of the TNT^® T7 Quick Coupled Transcription/Translation System in a process called the ‘ARM’ technique where antibody-ribosome-mRNA (ARM) complexes are selected by protein binding. The ARM complexes are generated during translation of mRNAs that lack stop codons. The functional antibody that is still complexed with the ribosome and mRNA is selected by protein-coupled magnetic beads, and the associated mRNA is reverse-transcribed and amplified by polymerase chain reaction (RT-PCR). The PCR products are then used in subsequent cycles of the method. (2016)

Notes: This Tech. Sight article describes the in vitro expression cloning technique (IVEC). The technique involves expression of plasmid pools (~50-100 clones/pool) in the TNT® Coupled Reticulocyte Lysate System, where approximately 30 proteins can be expressed in a single reaction. Each pool is screened for a certain biochemical activity, and positive pools are progressively subdivided until a single cDNA encoding the active protein is isolated. The technique is demonstrated diagrammatically with post-translational modifications (e.g., phosphorylation, degradation and cleavage of radiolabeled protein), but other activities could be assayed with this technique. (0901)

Notes: Glioma cell lines derived from glioblastoma tumors were rapidly screened for premature stop codon or frameshift mutations in the PTEN mRNA by the protein truncation test (PTT). PTT templates were generated by RT-PCR with a T7 RNA polymerase transcription site in the upstream primer and translated in the TNT^® T7 Quick Coupled Transcription/Translation System. (1131)

Notes: The cDNA for type IV adenylyl cyclase (ACIV) was expressed in the TNT^® Coupled Reticulocyte Lysate System in the presence of [³⁵S]methionine. Adenylyl cyclase activity was assayed, and the protein was immunoprecipitated with antibodies to ACIV. The adenylyl cyclase was activated by bovine brain Gs and forskolin and was quite stable. This report showed that ACIV can be produced in vitro with the activity of baculovirus-produced ACIV. (1777)

Notes: Different RD1-CAT [³⁵S]methionine-labeled chimeric proteins were produced using the TNT^® Coupled Reticulocyte Lysate System. The chimeric proteins showed that the unique N-terminal domain of RD1 confers membrane association upon the cytosolic CAT protein. (0419)

Notes: [³⁵S]-methionine-labeled pRB60, pRB50 and pRB105 proteins were synthesized using TNT® Coupled Reticulocyte Lysate System. Experiments were performed with these proteins and GST fusion proteins to determine protein-binding sites. At low levels of GST-fusion protein, the pRB60 protein bound immobilized GST-E7(3-98) protein but did not bind the GST-E7-CR3 fusion or GST. The other truncated pRB translation products did not bind any GST constructs. With higher E7 concentrations, all pRB products bound the full length GST-E7 and CR3 domain fusion protein but did not bind GST. Complete binding was not observed, suggesting that denaturation, phosphorylation or other modifications render some fraction of these proteins unable to bind. The E7 CR2 and CR3 domains bind to non-overlapping domains on pRB. (0002)