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Hepatology 27, 254-263. Hepatitis B virus with antigenically alter hepatitis B surface antigen is selected by high-dose hepatitis B immune globulin after liver transplantation 1998

Protzer-Knolle, U., Naumann, U., Bartenschlager, R., Berg, T., Hoff, U., Meyer zum Buschenfeldee, K.-H., Neuhaus, P., Gerken, G.

Notes: Several variant hepatitis B isolates were subjected to PCR to isolate the surface antigen cDNA. The PCR products were cloned and subjected to in vitro transcription/translation with the TNT® Coupled Reticulocyte Lysate System in the presence of Canine Pancreatic Microsomal Membranes (CMMs). The amount of microsomal membranes was adjusted to give the approximate ratios of wild-type glycosylated and unglycosylated forms. The variants were then tested for immunoprecipitation with a commonly used anti-hepatitis B surface antigen pAb. Only one of the variants was immunoprecipitated. (0520)

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Proc. Natl. Acad. Sci. USA 95, 8087-8092. Identification of a human PTS1 receptor docking protein directly required for peroxisomal protein import. 1998

Fransen, M., Terlecky, S.R., Subramani, S.

Notes: The human protein hsPex14p as welll as another protein hsPex13p and hsPex5p were cloned into the PinPoint® Xa2 vector and expressed in JM109 E.coli. The cell extracts were run on SDS PAGE gels, transferred to nitrocellulose and probed with the anti-MF3 antisera. The hsPex5p, supposedly the human PTS1 homolog, was translated in vitro with the TNT® Coupled Reticulocyte Lysate System and reacted with another blot with the same proteins and the hsPex14p bound the 35S-labeled protein. (1172)

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J. Biol. Chem. 273, 35008-35015. Molecular cloning and characterization of a novel retinoic acid-inducible gene that encodes a putative G protein-coupled receptor. 1998

Cheng, Y. , Lotan, R.

Notes: These authors used the TNT® T7 Coupled Reticulocyte Lysate System together with Canine Pancreatic Microsomal Membranes (CMMs) to show post-translational modification of RAIG1. They also used RQ1 RNase-free DNase to treat RNA samples prior to cDNA synthesis. (1337)

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J. Biol. Chem. 273, 21594-21602. Molecular cloning and characterization of a transcription regulator with homology to GC-binding factor 1998

Reed, A.L., Yamazaki, H., Kaufman, J.D., Rubinstein, Y., Murphy, B., Johnson, A.C.

Notes: The cloned 752 amino acid GC-binding factor 2 (GCF2) was expressed in vitro using the TNT® Coupled Reticulocyte Lysate System. The synthesized product migrated at ~160kDa by SDS-PAGE when the predicted molecular weight was 83kDa. Expression of the protein from E.coli also produced a protein that migrated at ~160kDa. Mass spectrum analysis confirm a mass of 83kDa. The E.coli produced protein was further characterized by gel shift analysis with the AP-2 Concensus Oligonucleotide. Recombinant GCF2, AP-2 and SP1 transcription factors were analyzed for binding to the EGF receptor promoter by footprint analysis. The GCF2 protein was expressed with a EGF Receptor promoter-CAT reporter vector in CV-1 cells and found to increase CAT activity. (0510)

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Am. J. Hum. Genet. 62, 269-277. Nearby stop codons in exons of the neurofibromatosis type 1 gene are disparate splice effectors. 1998

Hoffmeyer, S., Nurnberg, P., Ritter, H., Fahsold, R., Leistner, W., Kaufmann, D., Krone, W.

Notes: cDNA synthesis was performed in a reverse transcription reaction containing single-stranded binding protein and RNasin® Ribonuclease Inhibitor. In a protein truncation test (PTT), five overlapping segments amplified by RT-PCR of NF1 transcript were expressed by TNT® Coupled Reticulocyte Lysate System. (1051)

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Am. J. Hum. Genet. 63, 1294-1306. Phenotype-genotype relationships in complementation group 3 of the peroxisome-biogenesis disorders. 1998

Chang, C. C. , Gould, S. J.

Notes: The TNT® T7 Coupled Reticulocyte Lysate System was used to synthesize and label PEX12 proteins with [35S]methionine. (1360)

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J. Biol. Chem. 273, 30973-30978. Protein transport into 'complex' diatom plastids utilizes two different targeting signals. 1998

Lang, M., Apt, K.E., Kroth, P.G.

Notes: The authors used TNT® Coupled Reticulocyte Lysate Systems together with Canine Pancreatic Microsomal Membranes (CMM) to perform heterologous import experiments with trypsin protection of a diatom nuclear-encoded plastid protein. (0836)

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Mol. Cell. Biol. 18, 3245-3256. RACK 1, a receptor for activated C kinase and a homolog of the β subunit of G proteins, inhibits activity of Src tyrosine kinases and growth of NIH 3T3 cells 1998

Chang, B.Y., Conroy, K.B., Machleder, E.M., Cartwright, C.A.

Notes: The authors used a TnT® Coupled Reticulocyte Lysate System to transcribe and translate Src in vitro. Cell proliferation of NIH 3T3 cells that stably expressed either HA-RACK or vector only was assessed using the CellTiter 96® Non-Radioactive Cell Proliferation Assay. (2537)

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J. Biol. Chem. 273, 25654-25658. Regulation of DNA-dependent protein kinase by the Lyn tyrosine kinase. 1998

Kumar, S., Pandey, P., Bharti, A., Jin, S., Weichselbaum, R., Weaver, D., Kufe, D., Kharbanda, S.

Notes: The authors used Promega's purified DNA-Dependent Protein Kinase as a substrate for in vitro phosphorylation by the protein tyrosine kinase Lyn. They also used the TNT® Coupled Reticulocyte Lysate System to synthesize radiolabeled DNA-dependent protein kinase (DNA-PK) catalytic subunit polypeptides for protein:protein interaction studies with GST-tagged Lyn. (0863)

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J. Biol. Chem. 273, 8398-8406. Substitution of the degenerate smooth muscle (SM) α-actin CC(A/T-rich)6GG elements with c-fos serum response elements results in increased basal expression but relaxed SM cell specificity and reduced angiotensin II inducibility. 1998

Hautmann, M.B., Madsen, C.S., Mack, C.P., Owens, G.K.

Notes: BAEC (bovine aortic endothelial) cells were transfected using Transfectam® Reagent. Serum response factor (SRF) was synthesized in vitro using the TNT® Coupled Reticulocyte Lysate System. (1070)

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Am. J. Hum. Genet. 63, 749-759. Systematic analysis of hMSH2 and hMLH1 in young colon cancer patients and controls. 1998

Farrington, S. M. , Lin-Goerke, J. , Ling, J. , Wang, Y. , Burczak, J. D. , Robbins, D. J. , Dunlop, M. G.

Notes: cDNA was generated by reverse transcription of RNA purified from lymphoblastoid cell lines. PCR amplification of the cDNA was used to introduce a 17bp consensus T7 promoter sequence and a mammalian translation-initiation sequence in frame with a unique hMLH1 or hMSH2 sequence. Resultant PCR products were translated in the TNT® T7 Coupled Reticulocyte Lysate System. (1194)

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J. Biol. Chem. 273, 25880-25888. UDP-galactose:ceramide galactosyltransferase is a class I integral membrane protein of the endoplasmic reticulum. 1998

Sprong, H., Kruithof, B., Leijendekker, R., Slot, J. W., van Meer, G., van der Sluijs, P.

Notes: The authors used the TNT® T7 Coupled Reticulocyte Lysate System and Canine Pancreatic Microsomal Membranes (CMMs) to make UDP-galactose:ceramide galactosyltransferase (CGalT) to test antibodies for their ability to immunoprecipitate this protein. (0364)

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Proc. Natl. Acad. Sci. USA 94, 6216-6221. A new member of the tumor necrosis factor/nerve growth factor receptor family inhibits T cell receptor–induced apoptosis. 1997

Nocentini, G., Giunchi, L., Ronchetti, S., Krausz, L.T., Bartoli, A., Moraca, R., Migliorati, G. and Riccardi, C.

Notes: The TNT® Coupled Transcription/Translation System was used to verify the 25.3kDa size of the cloned receptor. AMV reverse transcriptase was used for first strand cDNA synthesis prior to PCR. (1520)

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J. Cell Biol. 137, 27-35. A role for the M9 transport signal of hnRNP A1 in mRNA nuclear export. 1997

Izaurralde, E., Jarmolowski, A., Beisel, C., Mattaj, I.W., Dreyfuss, G., Fischer, U.

Notes: Full-length, human hnRNP A1 protein and truncated hnRNP (lacking the M9 domain, A1DeltaM9) were synthesized using the TNT® Coupled Reticulocyte Lysate System. The synthesized proteins were injected directly into oocytes without purification and were assayed for nucleocytoplasmic transport. (0964)

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J. Biol. Chem. 272, 15993-16001. Activation transcription factor 1 involvement in the regulation of murine H-2Dd expression. 1997

Ishiguro, N., Brown, G.D., Meruelo, D.

Notes: pSV-β-Galactosidase Control Vector was used as a transfection control in F9 cells and TNT® T7 Coupled Reticulocyte Lysate System was used to translate ATF-1 protein for use in gel shift assays. (1004)

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Mol. Cell. Biol. 17, 1832-1839. AF-2 activity and recruitment of steroid receptor coactivator 1 to the estrogen receptor depend on a lysine residue conserved in nuclear receptors. 1997

Henttu, P.M.A., Kalkhoven, E. and Parker, M.G.

Notes: [35S]methionine-labeled RIP-140, SRC-1, TIF1 and TIF2 were produced using the TNT® Coupled Reticulocyte Lysate System. The labeled proteins were incubated with GST or GST-AF2 receptor fusion proteins coupled to glutathione beads in the absence or presence of oestradiol or 4-hydroxytamoxifen. After overnight incubation and washing away of free protein, bound proteins were extracted and separated by SDS-PAGE. From binding studies with wild type or mutant AF2 receptors, SRC-1, TIF1 and TIF2 require Lys-366 in AF-2 for their interaction, but RIP-140 does not. (1797)

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Mol. Cell. Biol. 17, 989-998. An alternative splicing event which occurs in mouse pachytene spermatocytes generates a form of DNA ligase III with distinct biochemical properties that may function in meiotic recombination. 1997

Mackey, Z.B., Ramos, W., Levin, D.S., Walter, C.A., McCarrey, J.R. and Tomkinson, A.E.

Notes: The DNA strand break repair protein encoded by the human XRCC1 gene was previously reported to interact with the DNA ligase III-alpha protein. An XRCC1 polypeptide was synthesized with [35S]methionine using the TNT® Coupled Reticulocyte Lysate System. GST-DNA ligase fusion proteins were adenylated, separated by denaturing gel electrophoresis and transferred to nitrocellulose. After renaturation, the membranes were incubated with the labeled XRCC1 polypeptide. XRCC1 formed a complex with DNA ligase III-alpha, but there was no detectable complex between XRCC1 and DNA ligase III-beta. (1770)

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Nucl. Acids Res. 25, 5132-5134. Antibody-ribosome-mRNA (ARM) complexes as efficient selection particles for in vitro display and evolution of antibody combining sites 1997

He, M. and Taussig, M.J.

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)

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Biochemistry 36, 1287-1294. Association of domains within the cystic fibrosis transmembrane conductance regulator. 1997

Ostedgaard, L.S., Rich, D.P., DeBerg, L.G., Welsh, M.J.

Notes: Cystic fibrosis transmembrane conductance regulator (CFTR) was expressed in the TNT® Coupled Reticulocyte Lysate System to confirm its molecular weight. Translation was performed with Canine Pancreatic Microsomal Membranes (CMMs) to verify that the protein is integrated into the membrane. Further, the electrophoretic mobility of CFTR suggests that it is glycosylated. (0570)

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Science 278, 294-298. Caspase-3-generated fragment of gelsolin: effector of morphological change in apoptosis. 1997

Kothakota, S., Azuma, T., Reinhard, C., Klippel, A., Tang, J., Chu, K., McGarry, T.J., Kirschner, M.W., Koths, K., Kwiatkowski, D.J., Williams, L.T.

Notes: This article details the use of the IVEC technique in the TNT® Coupled Reticulocyte Lysate System to screening 100,000 cDNA clones to identify gelsolin as a substrate of caspase-3 protease. (0885)

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J. Biol. Chem. 272, 31793-31800. CCAAT/Enhancer-binding protein delta activates insulin-like growth factor-I gene transcription in osteoblasts. Identification Of a novel cyclic amp signaling pathway in bone 1997

Umayahara, Y., Ji, C., Centrella, M., Rotwein, P., McCarthy, T.L.

Notes: Studies were performed in COS-7 cells. The experimental firefly luciferase vector (derived from pGL2-Basic Vector) was co-transfected with the Renilla control Vector (pRL-CMV Vector) and plasmids expressing either the CCAAT/Enhancer-binding protein beta or delta from a CMV promoter. The firefly luciferase:expression plasmid ratio was 10:1 and the firefly luciferase:Renilla luciferase vector ratio was 1000:1. In vitro translations were performed with the TNT® Coupled Reticulocyte Lysate System. (0252)

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Proc. Natl. Acad. Sci. USA 94, 4943-4947. Characterization and tissue-specific expression of the rat basic fibroblast growth factor antisense mRNA and protein. 1997

Knee, R., Li, A.W. and Murphy, P.R.

Notes: Promega's M-MLV Reverse Transcriptase, TNT® Coupled Reticulocyte Lysate System, and Taq DNA Polymerase were used in this study. (2000)

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J. Biol. Chem. 272, 11770-11777.. Characterization of the FET4 protein of yeast. Evidence for a direct role in the transport of iron. 1997

Dix, D. , Bridgham, J. , Broderius, M. , Eide, D.

Notes: Various portions of a 552 residue protein were cloned and expressed in BL21(DE3)pLysS using the pGEMEX®-1Vector. The resulting proteins were purified and used as immunogens. The TNT® Coupled Reticulocyte Lysate System was used for in vitro translation. (1236)

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Proc. Natl. Acad. Sci. USA 94, 7406-7411. Cloning of a gene (RIG-G) associated with retinoic acid-induced differentiation of acute promyelocytic leukemia cells and representing a new member of a family of interferon-stimulated genes. 1997

Yu, M., Tong, J-H., Mao, M., Kan, L., Liu, M-M., Sun, Y-W., Fu, G., Jing, Y-K.,Yu, L., Lepaslier, D., Lanotte, M., Wang, Z-Y., Chen, Z., Waxman, S., Wang, Y-X., Tan, J-Z. and Chen, S-J.

Notes: Promega's pGEM®-T Vector System, pCI Mammalian Expression Vector and TNT® Coupled Reticulocyte Lysate System were used in this study. (1951)

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J. Biol. Chem. 272, 30715-30723. Cotranslational membrane insertion of the serine proteinase precursor NS2B-NS3 (Pro) of Dengue virus type 2 is required for efficient in vitro processing and is mediated through the hydrophobic regions of NS2B. 1997

Clum, S., Ebner, K.E. and Padmanabhan, R.

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)

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