Notes: The authors developed an in vitro selection system that is based on ribosome display and favors identification of 5´-untranslated regions (UTRs) with high translation efficiencies. A 5´-UTR random library was created in which the 5´-UTRs were upstream of a polyhistidine-tag/Renilla luciferase-coding region. In vitro transcripts from this library were translated in vitro using the Flexi^® Rabbit Reticulocyte Lysate System. The authors preferentially selected mRNAs with high translational efficiencies by shortening the translation time and capturing ternary complexes of mRNA, ribosome and nascent proteins. These complexes were captured using MagneHis™ Ni Particles. RNA was extracted from these complexes and used as a template in RT-PCR for the next round of selection. Before and after each round of selection, 9µl of RNA was translated in vitro, and 20µl of translated product was removed every 5 minutes to measure Renilla luciferase activity and monitor translation efficiency. Renilla luciferase was measured using the Renilla Luciferase Assay System. After two rounds of selection, RT-PCR products were cloned into a pUC18 vector, the sequences of the resulting plasmids were confirmed, and 0.5µg of plasmid was translated in vitro using the TNT^® T7 Coupled Rabbit Reticulocyte Lysate System to further evaluate translation efficiency. (3963)

Notes: Authors used the Flexi^® Rabbit Reticulocyte Lysate System to in vitro translate Fli-1 mRNA. The lysate was programmed with mRNAs containing mutated sequences to determine the mechanism of translation initiation of two different isoforms. (2210)

Notes: Transcribed cDNA for type X collagen using Promega's T7 RNA Polymerase. Translated this using Flexi^® Rabbit Reticulocyte Lysate System in presence of semi-permeabilized HT1080 cells to allow for correct assembly of fibrillar collagens. (0714)

Notes: To produce site-specific photolabile ADP-ribosylation factor mutants, the amino acid of interest was mutated to the Amber stop codon in the cDNA. The cDNA was then in vitro transcribed and the resulting RNA translated in vitro with the Flexi^® Rabbit Reticulocyte Lysate System supplemented with a suppressor tRNA charged in vitro with the L-4'-(3-trifluoromethyl-3H-diazirin-3-yl)-phenylalanine. The suppressor tRNA was used at 5µM in the reaction. The reaction was performed with labeled methionine to judge the success of the suppression via autoradiography. The photolabile protein was used to crosslink to proteins interacting with ADP-ribosylation factor. (0057)

Notes: Inserts in the pSP72 Vector were in vitro transcribed and translated using the Flexi^® Rabbit Reticulocyte Lysate System. (0340)

Notes: The authors co-translated mRNAs for beta 2 microglobulin and MHC class I proteins in Flexi^® Rabbit Reticulocyte Lysate System with Canine Pancreatic Microsomal Membranes (CMMs). They generated mRNAs using RiboMAX™ T7 Large Scale RNA Production System. Treated transcription reactions with RQ1 RNase-Free DNase I after transcription to remove template DNA. (0393)

Notes: The Flexi^® Rabbit Reticulocyte Lysate System was used for cell-free translation reaction in the presence of 4 x 10⁵ digitonin-permeabilized HT-1080 fibroblasts. Two types VIII chains were expressed as well as the a1chain of type X collagen. The expressed proteins were assayed by protease digestion and thermal denaturation for their quaternary structure. The in vitro transcripts for the translation reaction were produced with the Riboprobe^® System. (0995)

Notes: Several different gap junction channel subunit isotypes, also known as connexins, were synthesized in vitro in the presence of Canine Microsomal Membranes. Translation reactions were spun through a sucrose cushion to separate the membrane fraction from the supernatant. Connexins that integrated into the microsomes formed homo- and heterooligomeric structures. The assembled proteins in the microsome were fused with a synthetic membrane and single channel conductance was measured and found to be very similar to wild-type membranes. (1618)

Notes: PolyA+ RNA from HL-60 cells was in vitro translated using the Rabbit Reticulocyte Lysate System in the presence or absence of Canine Microsomal Membranes, then immunoprecipitated and analyzed by SDS-PAGE and fluorography. The calreticulin protein was presumably glycosylated by the membranes. The same results were obtained with in vitro transcripts of the calreticulin. (1617)

Notes: Two site-directed mutants of KV1.4 were expressed in the Flexi^® Rabbit Reticulocyte Lysate System in the presence of Canine Pancreatic Microsomal Membranes (CMMs). The membranes were gently pelleted, resuspended and the proteins in vitro phosphorylated with Calmodulin-dependent protein kinase II. (0490)

Notes: To demonstrate an interaction between Frzb and Wnt proteins in vitro, the proteins were cotranslated using Rabbit Reticulocyte Lysate in the presence of Canine Microsomal Membranes then immunoprecipitated with specific antibodies to either one. Control immunoprecipitations were performed with cotranslations with Frzb and the β-lactamase control or Wnt and the β-lactamase control. The procedure for the immunoprecipitation is referenced. (1603)

Notes: The cRNA for the Stimulator of Fe Transport (SFT) was in vitro translated with Rabbit Reticulocyte Lysate in the presence of Canine Microsomal Membranes. The RRL was used at 70% of total volume and 2 equivalents of the membranes were used. After translation, the reaction was treated with RNase A to reduce background. The SFT protein is predicted to have six transmembrane domains. (1621)

Notes: Reactions were performed at 30°C in a 25µl volume with 3 equivalents of membranes. Following translation, the membranes were lysed with detergent and the proteins immunoprecipitated with various antisera. The immunoprecipitated proteins were also subjected to endoglycosidase H digestion. (1598)

Notes: Different truncations of the protein of interest were in vitro translated in the presence of the membranes. The translation reaction were centrifuged and the pellet and supernatants assays for the presence of the protein product. (1628)

Notes: The CD69 transcripts were prepared with the T3 RiboMAX® System and translated with the Flexi® System in the presence of the membranes. After 60 minutes at 30°C, the translation reaction was layered onto a sucrose solution and centrifuged at 100,000 x g for 20min. The supernatant was discarded and the pellet was resuspended in SDS-PAGE sample buffer or digested with endoglycosidase H. (1601)

Notes: The two products were used to show that the rat a7 cDNA was translated and could be processed in vitro to a higher molecular weight, most likely by glycosylation. (1630)

Notes: The proteins r1 and r2 as well as the N-terminal half of r1 and the C-terminus of r1 were expressed using the Flexi^® Rabbit Reticulocyte Lysate System in the presence of Canine Microsomal Membranes. The r1 protein has four membrane spanning domains. The orientation of the protein was determined by proteinase K digestions of intact membranes. Digestions were terminated by the addition of PMSF rather than direct lysis in SDS sample buffer. (1622)

Notes: The paper looked at the insertion of a model protein with one, two and four transmembrane segments and different distributions of positively charged residues in the N-terminal tail and the polar loops. The proteins were translated in the presence of the microsomes. Translocation of polypeptides to the lumenal side of the microsomes was assayed by prevention of N-linked glycosylation through competitive inhibition by the addition of a glycosylation acceptor tripeptide but not by a nonacceptor tripeptide and by proteinase K treatment of the microsomes. All techniques are referenced. (2033)

Notes: Increasing residues of the chloride channel were fused to a prolactin epitope and translated in vitro using Promega's Rabbit Reticulocyte Lysate in the presence of Canine Microsomal Membranes. The proteins were subjected to a proteinase K digest in the presence or absence of Triton X-100. The protease digestions were stopped with PMSF and the translation reaction analyzed by Western blotting. The protease protection assays were used to define the topology of the channel. The chloride channel crosses the membrane at least 10 times. Very good protocol for the protease protection assay. (1596)

Notes: The extracellular domain of the receptor was expressed in vitro using Promega's Rabbit Reticulocyte Lysate in the presence or absence of the microsomes. The 60kDa extracellular domain is processed to a 70kDa glycoprotein in the presence of the membranes. The paper also shows a titration of the membranes in the translation reaction from 1-4µl per reaction. More protein is processed with increasing amounts of the microsomes. (1627)

Notes: Truncation mutants of the protein of interest were translated in the presence of the membranes. The membranes were pelleted and analyzed directly or treated with endoglycosidase F. The supernatents from the centrifugation were acetone precipitated and analyzed as well. (1602)

Notes: The 3AB protein was fused with a FLAG tag. The rabbit lysate was centrifuged prior to use to remove any protein aggregates. The reaction was set up in a 200µl volume with the 3AB protein transcript and a b globin mRNA. The reaction was split into two 100µl reactions and to one 25 equivalents of membranes were added and to the other, just buffer. The translation reactions were allowed to procede for 30min at 30°C, then translation stopped by the addition of cycloheximide to 300µM. The reactions were centrifuged at through a sucrose gradient at 86,000 x g. Fractions were removed and subjected to immunoprecipitation and gel analysis. The 3AB protein was found to be membrane associated. Further studies with the addition of the Canine Microsomal Membranes after translation and cycloheximide demonstrated a membrane association after translation. (1599)

Notes: Translations with the Flexi Rabbit Reticulocyte Lysate System were performed in 25µl volume containing 3 equivalents of membranes. Reactions were performed at 28.5°C and in the presence or absence of the glycosylation inhibitor AcNYT. Translation products were analyzed either directly by SDS-PAGE followed by fluorography or with prior immunoprecipitation of the translation products. Capped transcripts were produced by in vitro transcription with SP6 RNA Polymerase. (2029)

Notes: The novel UT1 urea transporter was translated in vitro in the presence or absence of the microsomes. In the absence of the microsomes, the protein migrates at 76kDa and in the presence, 86kDa. The data is not shown but described in the text. (1597)

Notes: The RSP2 protein was in vitro translated using Promega's Rabbit Reticulocyte Lysate in the presence of microsomes. The protein was not protected from proteinase K digestion and deemed to be a cytoplasmic protein. (1626)