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| 1. |
Hurst, R., Hook, B., Slater, M.R., Hatrnett, J., Storts, D.R., and Nath, N.
(2009)
Protein-protein interaction studies on protein arrays: effect of detection strategies on signal-to-background ratios.
Anal Biochem.
392
,
45-53
.
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Notes:
These authors compared 6 different detection strategies for protein-protein interactions on protein arrays. They expressed HaloTag® labeled bait proteins in a cell-free expression system, and captured these bait proteins onto coated glass slides using the HaloLink™ Array System. They then compared detection strategies using prey proteins labeled as follows: 1)35S methionine, 2) fluorescence (BODIPY-FL) and 3) biotin labeling of lysine residues using modified Lys tRNA, 4) chemical labeling after expression, 5) HaloTag® fusion, and 6) N-terminal FLAG tag. The authors evaluated signal:background ratios, adaptability to high-throughput screening, and ease of use.
(0003999) |
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Products: FluoroTect™ GreenLys in vitro Translation Labeling System | HaloLink™ Array (TNT® SP6 Wheat Germ) Two Slide System | HaloTag® Biotin Ligand | HaloTag® TMR Ligand | pF1A T7 Flexi® Vector | pFN19K HaloTag® T7 SP6 Flexi® Vector | TNT® SP6 Quick Coupled Transcription/Translation System, Trial Size | Transcend™ tRNA |
| 2. |
Shibuya, N., and Nakashima, N.
(2008)
Characterization of the 5' internal ribosome entry site of Plautia stali intestine virus.
J. Gen. Virol.
87
,
3679-3686
.
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Notes:
The Plautia stali virus contains two open reading frames and includes a 5´ internal ribosome entry site (IRES) and an intergenic IRES region. These authors showed that the 5´ IRES was functional and initiated translation in insect cell lysate, but not in rabbit reticulocyte lysate or wheat germ extract. The efficiency of translation mediated by the 5´ IRES region was tested with and without cap analogue using various firefly and Renilla luciferase reporter constructs. They also used deletion mutants to identify the specific regions required for translation initiation.
(0003942) |
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Products: FluoroTect™ GreenLys in vitro Translation Labeling System | pSP-luc+NF Fusion Vector | Transcend™ tRNA |
| 3. |
Nakajima, D., Saito, K., Yamakawa, H., Kikuno, R.F., Nakayama, M., Ohara, R., Okazaki, N., Koga, H., Nagase, T. and Ohara, O.
(2005)
Preparation of a Set of Expression-Ready Clones of Mammalian Long cDNAs Encoding Large Proteins by the ORF Trap Cloning Method.
DNA Research
12
,
257–267
.
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Notes:
To prepare expression-ready cDNA clones of 1589 putative full-length ORFs (from human and mouse genes) with an average size of 2.8 kb, a linear trap vector was created. Generated by PCR, this linear plasmid contained gene-specific sequences to allow homologous recombination. In addition, 5–10µg of a plasmid containing the same gene-specific sequence and the linear trap vector were purified using the Wizard® SV 96 PCR Clean-Up System. The purified DNA was resuspended in water and transformed into E. coli cells. The plasmid purified after the recombination was transferred to a Gateway expression vector and 100–200ng expressed in the TNT® T7 Quick Coupled Reticulocyte Lysate System with 0.2µl of FluoroTect™ GreenLys tRNA. The proteins expressed were resolved using SDS-PAGE.
(0003439) |
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Products: FluoroTect™ GreenLys in vitro Translation Labeling System | TNT® T7 Quick Coupled Transcription/Translation System | TNT® T7 Quick Coupled Transcription/Translation System, Trial Size | Wizard® SV 96 PCR Clean-Up System |
| 4. |
Talmud, P.J., Palmen, J., Putt, W., Lins, L., and Humphries, S.E.
(2005)
Determination of the functionality of common APOA5 polymorphisms.
J. Biol. Chem.
280
,
28215-28220
.
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Notes:
The authors investigated common variants of the APOA5 gene that have been associated with differences in plasma triglyceride (TG) levels. PCR fragments containing either the –1131T --> C promoter variant or containing both the –1131T --> C and –3G --> A promoter variants were cloned into the pGEM®-T Vector System. The fragments were subsequently cloned into the pGL3 Basic Vector and transiently transfected into Huh7 and HepG2 cells along with the luciferase control vector, pRL-TK. The cells were lysed 48 hours after transfection and Luciferase activity was measured with the Dual-Luciferase® Reporter Assay System. The function of the 1891T --> C variant in the 3´ UTR was tested the same way; with the exception that site-directed mutagenesis was performed to introduce the T --> C at position 1891 before the fragment was cloned into the pGL3 Basic Vector. The functionality of the Kozak sequence –3A --> G variant was determined by cloning cDNAs into the pGEM®-7Zf Vector. Transcription/translation experiments were performed using the TNT® Quick Coupled Transcription/Translation System and the proteins were labeled using the FluorTect™ GreenLys System. In addition, a primer extension inhibition assay was performed using capped mRNAs generated with the Riboprobe® System –T7 and the Ribo m7G Cap Analog. Ribosome binding reactions were performed using the Rabbit Reticulocyte Lysate System, Nuclease Treated.
(0003460) |
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Products: Dual-Luciferase® Reporter Assay System | Dual-Luciferase® Reporter Assay System 10-Pack | FluoroTect™ GreenLys in vitro Translation Labeling System | pGEM®-7Zf(–) Vector | pGEM®-7Zf(+) Vector | pGEM®-T Vector System I | pGEM®-T Vector System II | pGL3-Basic Vector | Rabbit Reticulocyte Lysate System, Nuclease Treated | Ribo m7G Cap Analog | Riboprobe® System—T7 | TNT® T7 Quick Coupled Transcription/Translation System | TNT® T7 Quick Coupled Transcription/Translation System, Trial Size |
| 5. |
Suzuki, T., Kasahara, M., Yoshioka, H., Morohashi, K. and Umesono, K.
(2003)
LXXLL-related motifs in Dax-1 have target specificity for the orphan nuclear receptors Ad4BP/SF-1 and LRH-1.
Mol. Cell. Biol.
23(1)
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238-249
.
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Notes:
In this study, the authors investigated the interaction between the orphan receptors Dax-1 and Ad4bp/SF-1. Specific interaction was detected when the two proteins were used in both yeast and mammalian two-hybrid systems. To further characterize this interaction, an in vitro pull-down assay was performed. Dax-1 protein or a dax-1 mutant lacking an LXXLL motif were transcribed and translated in vitro using the TNT® T7 Quick System and Fluorotect™ GreenLys BODIPY labeled tRNA. The fluorescently labeled protein interacted with a Ad4BP/SF-1-maltose binding protein (MBP) fusion protein immobilized to an amylose resin. After elution and SDS-PAGE, the fluorescently labeled protein was detected using a Hitachi FMBIO® II Fluorescence Imaging System. The interaction between Dax-1 and Ad4BP/SF-1 was dependent on the LXXLL motif.
(0003237) |
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Products: FluoroTect™ GreenLys in vitro Translation Labeling System | TNT® T7 Quick Coupled Transcription/Translation System | TNT® T7 Quick Coupled Transcription/Translation System, Trial Size |
| 6. |
Doi N., Takashima H., Kinjo M., Sakata K., Kawahashi Y., Oishi Y., Oyama R., Miyamoto-Sato E., Sawasaki T., Endo Y. and Yanagawa H.
(2002)
Novel fluorescence labeling and high-throughput assay technologies for in vitro analysis of protein interactions.
Genome Res.
12(3)
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487-92
.
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Notes:
Template DNAs encoding c-Fos (118–211 amino acids) and c-Jun (216–318 amino acids) were generated by PCR, purified and then in vitro transcribed using the RiboMAX™ Large Scale RNA Production System. Following transcription, the RNA was purified and then translated in a wheat germ extract system supplemented with FluoroTect GreenLys tRNA. The fluorescently labeled proteins generated by the translation reaction were run on a 16.5% Tricine-SDS–PAGE and analyzed with a Molecular Imager FX. Labeling efficiency was calculated by measuring total protein via T7-antibody and determining the amount of fluorescently-labeled protein by fluorescence correlation spectroscopy (FCS).
(0003084) |
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Products: FluoroTect™ GreenLys in vitro Translation Labeling System | RiboMAX™ Large Scale RNA Production System—SP6 | RiboMAX™ Large Scale RNA Production System—T7 |
| 7. |
Iborra, F.J., Jackson, D.A. and Cook, P.R.
(2001)
Coupled transcription and translation within nuclei of mammalian cells.
Science
293(5532)
,
1139-1142
.
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Notes:
To understand if protein translation occurs in the nucleus of eukaryotic cells, the researchers in this study labeled proteins in vivo by bathing permeablized cells in a translation mix containing radiochemical, biotin or fluorescently tagged amino acids. The study found that all three types of label gave similar results. To fluorescently tag proteins, cells were grown, then permeablized, and bathed in a mix of buffer, creatine phosphokinase, phosphocreatine, GTP, tRNA, aminoacyl-tRNA synthetase, MgCl2, protease inhibitors, a 1:10 dilution of Fluorotect Greenlys BODIPY labeled lysine-tRNA complex and 50μM amino acid mixture minus lysine. The study revealed that, contrary to previous beliefs, some proteins are transcribed and translated within the nucleus in a coupled manner.
(0002744) |
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Products: FluoroTect™ GreenLys in vitro Translation Labeling System |