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. (3999)

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. (3942)

Notes: The MagneGST™ Protein Purification System was used to purify GST fusion proteins of the oncoprotein HPV16 E7 or various mutants of HPV16 E7. The purified GST fusion proteins were used for in vitro binding experiments with steroid receptor coactivator 1 (SRC-1), which was produced using the TNT^® T7 Coupled Wheat Germ Extract System and labeled with the Transcend™ Non-Radioactive Translation Detection System. GST pull-down assays were resolved by Western analysis using streptavidn-horseradish peroxidase and alpha-GST. To determine the effects of endogenously expressed HPV16 E7 on SCR-1-mediated transcription, luciferase reporters under the control of either the IL-8 promoter or an artificial promoter containing three estrogen response elements repeats (3 × EREs) were cotransfected with a Renilla control vector into two human cervical cancer lines (C33A and CaSki) using either the Transfast™ Transfection Reagent or another commercial transfection reagent. The Dual-Luciferase^® Reporter Assay was then used to determine luciferase activity to functionally map the E7-interacting domain and to determine the effects of high- and low-risk PHV E7s on SRC-1-mediated transcription. (3459)

Notes: The authors performed a yeast two-hybrid screen using big mitogen-activated kinase 1 (BMK1/ERK5) as the bait and identified the scaffolding protein 14-3-3beta. To confirm this interaction, the cloned mouse BMK1 gene was expressed in the TNT^® T7 Quick Coupled Transcription/Translation System. The expressed protein was labeled with Transcend™ tRNA.  Using a GST-14-3-3beta fusion protein, a pull-down assay was performed and the direct binding confirmed after immunoblotting and staining with streptavidin-horseradish peroxidase (HRP).  The interaction of various BMK1 mutants were tested in a mammalian two-hybrid system and measured by the Dual-Luciferase^® Reporter Assay System. (3078)

Notes: The sterol regulatory element-binding protein-1a (SREBP1a) was in vitro transcribed and translated from a plasmid template using TNT^® T7 Quick for PCR DNA. A control reaction was performed using Transcend™ tRNA to confirm that the 120KDa protein was expressed correctly.  Unlabeled SREBP1a was used in gel-shift assays with a labeled oligo representing the SRE motif from the human ABCD2 gene promoter.  (3222)

Notes: This paper describes using the TNT^® T7 Coupled Reticulocyte Lysate System and Transcend™ tRNA to perform a nonradioactive in vitro Protein Truncation Test for a BRCA2 gene product.  Templates for the transcription-translation reactions were created by PCR-amplifying regions of the BRCA2 gene from normal and cancer patient sample genomic DNA.  (3075)

Notes: The TNT® T3 Coupled Reticulocyte Lysate System was used to express the PTTG-binding factor, a 179 amino acid protein (predicted 19.7kDa). The protein was translated in the presence of the Transcend™ Biotinylated tRNA. The expressed protein was used to GST-fusion protein pulldown reactions with a PTTG-GST fusion. The bound proteins were removed by boiling in SDS sample buffer, electrophoresed, transferred, and detected by chemiluminescent HRP. (0032)

Notes: Various Stat-1 and IRF-1 proteins were expressed in vitro with the TNT® T7 Coupled Reticulocyte Lysate System for various protein-protein interaction studies by co-immunoprecipitation with antibodies followed by immunoblotting. To insure equal amounts of proteins were expressed and intact, the Transcend™ Biotinylated tRNA was incorporated during the reaction and a portion of the reaction was removed, electrophoresed, transferred and detected with an HRP-conjugate. (0030)

Notes: The 1736 amino acid (predicted MW of 194kDa) protein ZO-1 was translated in vitro with the TNT® T7 Coupled Reticulocyte System and the Transcend™ Biotinylated tRNA. There were 107 lysines in the protein. The protein was used in protein-protein interaction studies. A protein, JAM, was expressed as a GST fusion in E. coli and immobilized to a glutathione-linked sepharose beads or peptides were immobilized to sepharose beads. The translation extract was interacted with the beads, washed and bound protein removed by boiling in SDS sample buffer. The bound protein was electrophoresed, transferred and detected via the biotin tag on the ZO-1 protein. (0031)

Notes: The TNT® Coupled Reticulocyte Lysate System was used in conjunction with the Transcend™ Chemiluminescent Non-Radioactive Translation Detection System. Various truncations of the protein of interest were produced ranging from 67kDa to 19kDa to use in gel shift assays. The truncations were used to identify which region bound the oligo of interest. The in vitro translated proteins were also used with antibodies for supershifts. (0003)

Notes: A clone of the rat AF-9 cDNA was isolated. Two potential start codons were present in the cDNA. To see which is preferred by a mammalian translation system, the cDNA was translated in vitro with the TNT® Coupled Reticulocyte Lysate System and the Transcend™ Biotinylated tRNA. The preferred transcript was the first ATG resulting in the 370 amino acid (40.7kDa predicted molecular weight) which had 47 lysines. (0033)

Notes: The cDNA of the pectin methyltransferase was translated in vitro with the TNT® Coupled Reticulocyte Lysate System in the presence of the Transcend™ Biotinylated tRNA. The protein was used for in vitro enzymatic assay of activity. The assay was colorimetric and the heme in the rabbit lysate interfered. The hemoglobin was removed by acid precipitation and the method is referenced. The enzyme contains 555 amino acids and 39 lysines. (0029)

Notes: The authors used the TNT^® Coupled Reticulocyte Lysate System with Transcend™ Biotinylated tRNA to synthesize S10 and HSJ2 protein for protein:protein interaction studies with GST-tagged PTTG. (0556)

Notes: The proteins Ibp2, MP90 and luciferase were translated in vitro with the TNT^® Coupled Reticulocyte Lysate System in the presence of the Transcend™ Biotinylated tRNA. The translated proteins were reacted with a glutathione bead-immobilized portion of the clathrin heavy chain. Bound proteins were solubilized in SDS sample buffer and the proteins detected by Western detection with the Transcend™ Non-Radioactive Translation Detection System. (0990)

Notes: PEA-15 cDNAs were cloned from a mouse astrocytic library, and the clones were linearized and expressed using T3 or T7 Polymerase in the TNT^® Reticulocyte Lysate System either in the presence of [³⁵S]methionine or Transcend™ tRNA. The resulting proteins were analyzed by SDS-PAGE and Western blotting, respectively. A unique 15kDa protein that was recognized by an antibody to PEA-15 was produced in the TNT^® System. (1178)

Notes: In this work, Sanford and colleagues study the GTP-binding protein, Rab5, by expressing this protein in Rabbit Reticulocyte Lysate (RRL) with Transcend™ Biotinylated tRNA. In RRL, prenylated biotin-Rab5 associates with a 45kDa GDP dissociation inhibitor in a ~70kDa particle on sucrose density gradients. The authors suggest that biotin-Rab5 provides a novel tool for capturing and characterizing accessory factors required for Rab protein function. (0436)