Notes: Authors used the Rabbit Reticulocyte Lysate System to investigate the interaction between transcription and translation directed by poliovirus. Thirty-five microliters of lysate were supplemented with 4µg of a poliovirus-infected S10 HeLa extract containing the partially purified viral polymerase, and translation was monitored using a poliovirus replicon driving luciferase expression. RNA synthesis was measured by incorporation of ³²P-UMP into the poliovirus RNA. When ribosomes were actively translating, RNA synthesis was undetectable, but when cycloheximide was added to the reactions, the viral polymerase was able to incorporate the labeled nucleotide. (2172)

Notes: The authors incubated Canine Pancreatic Microsomal Membranes (CMMs) with various caspases to show cleavage of SRP 72. The treated membranes were incubated with β-lactamase made in Rabbit Reticulocyte Lysate and the effect of cleavage of SRP 72 on processing/transport of β-lactamase was evaluated. (0210)

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: The Quantilum™ Recombinant Luciferase was heat denatured in the presence of heat shock protein hsp 18.1 and the renaturation of the protein was assayed in with the Luciferase Assay System. The denaturation was performed in either Rabbit Reticulocyte Lysate or Wheat Germ Extract in the presence or absence of added ATP. (0809)

Notes: The AP-1 and CREB Consensus Oligonucleotides were used for gel shift assays with transfected HepG2 cell extracts, E.coli extracts containing eukaryotic transcription factors and a truncated c-jun protein expressed with Rabbit Reticulocyte Lysate. (1343)

Notes: Luciferase reporter constructs containing either the luc or luc+ gene (from pSP-luc+NF Fusion Vector) were used in in vitro transcription to produce capped mRNA. Luciferase was produced by in vitro translation using either neurospora extract or Promega's Rabbit Reticulocyte Lysate, Nuclease Treated and detected using the Luciferase Assay Reagent. (0208)

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: Transcripts were produced using the RiboMAX™ Large Scale RNA Production System and expressed in the Rabbit Reticulocyte Lysate System with or without Canine Pancreatic Microsomal Membranes (CMMs). Proteinase K protection assays were performed with proteins expressed in the presence of the membranes with and without Triton^® X-100 solubilization of the membranes. (1509)

Notes: The ribonuclease inhibitor was used to protect RNA during the reverse transcriptase reaction for RT-PCR. The RNasin® Ribonuclease Inhibitor is also used in the RiboMAX® System for RNA stabilization during in vitro transcription. Transcripts were produce with the RiboMAX® System and expresed in Rabbit Lysate with or without Canine Microsomal Membranes. Proteinase K protection assays are performed with proteins expressed in the presence of the membranes with and without Triton X-100 solubilization of the membranes. The proteinase K digests were stopped by the addition of PMSF and immediately loading the sample into 100°C SDS Sample buffer. (1670)

Notes: The pCI-neo Mammalian Expression Vector was used to construct a chimeric vector with the neo resistance gene downstream of an internal ribosome entry site, so that the neo gene was transcribed with the Cig30 cDNA and both protein controlled by the CMV promoter. The construct was used to make stable transfectants of the HIB-1B hibernoma cells. Cell lines were analyzed by Northern blotting. The protein was also analyzed in vitro with the Rabbit Reticulocyte Lysate and Canine Pancreatic Microsomal Membranes (CMMs). (0246)

Notes: All cDNAs to be translated in vitro were subcloned into the pSP64 Poly(A) Vector. The vector was linearized and RNA transcribed from the SP6 promoter with the Riboprobe^® in vitro Transcription System. The in vitro transcribed RNA was translated in the Rabbit Reticulocyte Lysate in the presence or absence of Canine Pancreatic Microsomal Membranes (CMMs). N-linked glycosylation sequences were engineered into the proteolipid protein to determine the orientation of the domains of the protein in the membrane. (1114)

Notes: ENC-1 and a T7 tagged ENC-1 were translated in the Rabbit Reticulocyte Lysate System to confirm the size of the protein products. (1039)

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: The authors prepared their own rat liver microsomes. Proteins were translated using Promega's Rabbit Reticulocyte Lysate in the presence or absence of the microsomes. The translated proteins were assayed for proteinase K protection in the presence or absence of Triton X-100. The proteinase K digests were stopped by the addition of PMSF. (1595)

Notes: This article describes an in vitro assay to activate hepatitis C virus (HCV) NS2-3 protease posttranslationally and methods studying the effects of several common inhibitors on enzymatic activity. HCV N2-3 protease was expressed using the Rabbit Reticulocyte Lysate System in the presence of [³⁵S]methionine, and the NS2-3 protease was activated by detergent cleavage. The protein was incubated with various proteinase inhibitors (aprotinin, antipain, DTT, E64, pepstatin, EGTA, EDTA, iodoacetamide, N-ethylaleimide, 1,10-phenanthroline, phenylmethylsulfonyl fluoride, soybean trypsin inhibitor, TLCK and TPCK). (0533)

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 authors used Promega's Canine Pancreatic Microsomal Membranes (CMMs) in combination Promega's Rabbit Reticulocyte Lysate to study the effects of various N-linked glycosylation mutations for TM mapping. Researchers included the detergent octaethylene glycol mono n-dodecyl ether from Nikkol in reactions without the CMMs to prevent aggregation of the proteins. (0541)

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 SILVER SEQUENCE™ DNA Sequencing System, Rabbit Reticulocyte Lysate System, Wizard^® Plus Minipreps DNA Purification System and Taq DNA polymerase were used in this study. (1473)

Notes: Covalent fusions of mRNA and the peptide or protein that it encodes were generated by in vitro translation in Rabbit Reticulocyte Lysate of mRNAs carrying puromycin. Puromycin, which is a peptidyl acceptor antibiotic, mimics the aminoacyl end of tRNA, enters the ribosomal A site and accepts the nascent peptide. Specific fusions of stable mRNA-peptide fusions were then selected from a pool of random sequence mRNA-peptide fusions by immunoprecipitation. This technique suggests a route for selection and evolution of proteins. (0484)

Notes: The authors used the Rabbit Reticulocyte Lysate together with Canine Pancreatic Microsomal Membranes (CMMs) to study the insertion of internal transmembrane domains. They removed ribosomes from Rabbit Reticulocyte Lysate by centrifugation and replaced with ribosomes isolated from a Wheat Germ Extract and restored functional translation with resulting Rabbit Reticulocyte Lysate supernatant (ribosome-free) fraction. (0196)

Notes: Promega's Casein Kinase II and DNA-Dependent Protein Kinase were used for in vitro phosphorylation of the SV40 large T antigen expressed either from Rabbit Reticulocyte Lysate or a large Tantigen-β-Gal fusion protein expressed in a rat hepatoma cell line. Lysates and cell extracts were assayed for DNA-dependent protein kinase activity with a peptide substrate. The ability of β-Gal-T antigen fusions (± phosphorylation) to bind to murine importin subunits was assessed by an ELISA system using the Anti-β-Galactosidase mAb. (0120)

Notes: The AMV Reverse Transcriptase, RNasin^® Ribonuclease Inhibitor and Taq DNA Polymerase were used for RT-PCR to isolate a clone of the human insulin upstream factor 1, a transcription factor. The protein was expressed in vitro with the Rabbit Reticulocyte Lysate and used for gel shift assays. Reporter studies were performed in MIN6 cells. (0762)

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)