Notes: The NS2B-NS3 protein was translated in the presence of the microsomes. Cotranslational insertion into the membrane was necessary for full processing of the precursor. As more membranes were added to the reaction, more protein was processed. Incubation of the in vitro reaction after the addition of cycloheximide with the microsomal membranes did not result in processed protein. (1650)

Notes: The TCRa was translated using the TNT^® Coupled Reticulocyte Lysate System in the presence or absence of canine pancreatic microsomes and demonstrated glycosylation of the protein. The researchers used their own preparation of microsomes made by the Walter and Blobel Protocol in Methods in Enzymology, Vol. 96, p84-93 (1983). (2045)

Notes: The Altered Sites^® II in vitro Mutagenesis System was used to produce several vitamin D receptor truncation mutants. The mutants were generated by the insertion of premature stop codons. The single-stranded protocol was used for the synthesis reaction and the newly synthesized DNA was propagated in BMH 71-18 mutS cells followed by growth in JM109 cells. Both mutant and wildtype receptor were cloned into pGEM^®-4 Vector and in vitro expressed with the TNT^® Coupled Reticulocyte Lysate System. The various forms of the receptor were assessed for protease sensitivity in the presence or absence of ligand. (0783)

Notes: Promega's pGEM®-T Vector System and TNT® Coupled Reticulocyte Lysate System were used in this study. (1948)

Notes: CAS and importin beta were cloned into the T7 60 Vector (Görlick et al. 1997), which contains a T7 promoter. The proteins were synthesized in the TNT® T7 Coupled Reticulocyte Lysate System with [35S]methionine label. The [35S]methionine-labeled proteins were incubated with importin alpha and forms of Ran. Permanently GTP-bound mutants of Ran abolish importin beta binding to alpha, but stimulate CAS-importin alpha interaction. (1824)

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: This research was based upon the technique of Powell et al. (1993) to screen for APC mutations in Ashkenazi Jews. A mutation of APC, which carries a T-A transversion but does not have any truncating mutations, causes truncations of translation in the TNT^® T7 Coupled Reticulocyte Lysate System. (0830)

Notes: The TNT® Coupled Reticulocyte System and Canine Pancreatic Microsomal Membranes (CMMs) were used to express a clone isolated through a differential display screen. (1291)

Notes: The Altered Sites® II in vitro Mutagenesis System was used to produce 27 mutant protein cDNAs. Some had up to three amino acid changes and one had a three amino acid deletion. All mutants were expressed in a TNT® Coupled Reticulocyte Lysate System reaction and the labeled proteins were used to demonstrate interactions with cyclin D1 and p16 by immunoprecipitation. (1315)

Notes: The TNT^® Coupled Reticulocyte Lysate System was used to characterize the membrane domain of several polytopic proteins. The two systems employed here use the presence or inhibition of glycosylation as an indicator of membrane insertion. The addition of a core glycosylation site resulted in an increase of ~2.5kDa to the relative molecular mass determined by SDS-PAGE. Using fusion vectors HK-M0 and HK-M1, the ability of each transmembrane domain to insert into the membrane was assessed. Of seven putative membrane segments, some had signal anchor properties, one had stop transfer properties and another had no membrane insertion activity. Combining data from the different systems confirmed seven transmembrane segments. (1649)

Notes: The Altered Sites^® II in vitro Mutagenesis System was used to introduce a point mutation D359E into the MDM2 oncoprotein. Wild-type MDM2 was in vitro translated with TNT^® T7Coupled Reticulocyte Lysate System and assayed for proteolysis by normal and apoptotic cell extracts. (1222)

Notes: [³⁵S]methionine-labeled pre-IL-1β and PARP(T) proteins were synthesized using the TNT^® T7 Coupled Reticulocyte Lysate System and were used for cleavage studies. The proteins were incubated with purified recombinant human ICE, which cleaved both proteins. The cleavage of PARP requires 50- to 100-fold higher concentrations of ICE than required for pre-IL-1β cleavage. (1060)

Notes: The TNT^® Coupled Rabbit Reticulocyte Lysate System was used for invitro translation of protein from a cDNA clone. This in vitro translated protein was used for gel shift assays and Scatchard analysis of the DNA-TR4 protein interaction. The pCAT^® Promoter Vector was used to assay the enhancer activities of the intronic region. (1537)

Notes: The TNT® Coupled Reticulocyte Lysate System was used to generate ³⁵S-labeled Staufen protein. The protein was used to demonstrate direct interaction between Staufen and the Inscrutable-GST fusion protein immobilized on glutathione beads. (1516)

Notes: The authors used the Promega pSV-β-Galactosidase Control Vector, pGL3-Control and pGL3-Basic Vectors, Luciferase Assay Reagent, TNT^® Coupled Wheat Germ Extract Systems and TNT^® Coupled Reticulocyte Lysate Systems in their studies NF-κB. (0994)

Notes: Poly(A+) RNA was isolated from total RNA of various rat tissues. Poly(A+) RNA was reverse transcribed with the RT System and the products used for 3'RACE. The TNT^® System was used to confirm the size of the expected protein. (2140)

Notes: Wildtype mouse oestrogen receptors and various tyrosine-replacement mutants were reacted with a biotinylated oestrogen response element and immobilized on the Streptavidin MagneSphere® Paramagnetic Particles (SA-PMPs). The immobilized complexes were then assayed for the ability to interact with in vitro translated RIP140 and SRC-1 protein in the presence or absence of 17β-estradiol. The capture proteins were analyzed by SDS-PAGE and fluorography. In vitro translations were performed with the TNT® Coupled Reticulocyte Lysate System. (0181)

Notes: Calf Intestinal Alkaline Phosphatase was used for in vitro dephosphorylation of the RAP46 protein, and the dephosphorylated protein was assayed for interaction with various transcription factors. The TNT^® Coupled Reticulocyte Lysate System was used to produce the transcription factors. The Rabbit Reticulocyte Lysate was used to aid protein refolding of heat denatured luciferase and the extent of refolding was examined with the Luciferase Assay System. (0084)

Notes: Three isoforms of the actin-binding LIM protein were expressed in vitro using the TNT^® Coupled Reticulocyte Lysate System in the presence of Canine Microsomal Membranes. The expressed proteins were used in an actin binding assay. (1631)

Notes: The AQP8 protein was expressed in vitro using the TNT^® Coupled Reticulocyte Lysate System in the presence of Canine Microsomal Membranes. The protein demonstrated an increase in molecular weight in the presence of the membranes. Promega's pGEM^®-T Vector System and Proteinase K were also used. (1624)

Notes: Point mutations in the repeated portion of the PKD1 gene were detected by the Protein Truncation Test. PCR products containing the T7 promoter were used as templates and protein was labeled by incorporation of [³H] leucine in a transcription/translation reaction using TNT^® Coupled Reticulocyte Lysate System. The pGEM^®-T Vector System was also used. (0489)

Notes: The NMDA subunit 2B (NR2B) and subunit 2A (NR2A) were in vitro transcribed/translated to test for specificity of an antibody raised to NR2B. The antibody precipitated the 162kDa NR2B protein but not the NR2A protein. The TNT^® Coupled Reticulocyte Lysate System was used for transcription/translation. (1525)

Notes: In order to locate the region containing the 10kDa protein, proteins encoded by the IBV sequence from 10752-12600 were transcribed and translation using the TNT^® T7 Coupled Reticulocyte Lysate System and immunoprecipitated with the V47 antiserum. Four protein species of approximately 38, 56, 60 and 70kDa were immunoprecipitated by antiserum V47. The results demonstrate that the antigenic determinant is encoded between nucleotides 11488 and 11739. (1767)

Notes: To search for mutations in the PTEN gene, PTT was performed using the TNT^® T7 Coupled Reticulocyte Lysate System, on 20 breast, 6 glioblastoma and 2 prostate tumor cell lines. Two truncating mutations were identified in PTEN in the breast samples, three in the six glioblastoma lines and in both of the prostate cancer cell lines. One of the mutations in the prostate cancer cell line affected translation initiation. It is believe that more mutations may exist in these lines but were not detected due to the limits of PTT and the fact that the entire coding region was not analyzed. (0794)

Notes: The pCI Mammalian Expression Vector was used to express the p64H1 protein in 293 cells and HT-4, a clonal neuronal cell line. Inserts in the vectors were verified for expression using the TNT^® Coupled Reticulocyte Lysate System. The proteins were translated in the presence of Canine Pancreatic Microsomal Membranes (CMMs). The pGEM^®-T Vector System was used to clone the PCR product of circle rapid amplification of cDNA ends to find the true 5'-end of the p64H1 cDNA. The Tfx™-20 Reagent was used to transfect 293 cells and HT-4 neuronal cells with the p64H1-expressing pCI-based plasmid. (1206)