Notes: These authors showed that double-substitution mutation of two N-terminal juxtaposed residues (from positive to neutral charged species) resulted in a reversal of the transmembrane orientation of the protein of interest. For in vitro transcription/translation, they used the TNT^® T7 Coupled Reticulocyte Lysate System in the presence of Canine Pancreatic Microsomal Membranes. A protease protection assay and an epitope-specific pull-down assay were used to determine the membrane orientation of the in vitro synthesized protein. (3051)

Notes: In this study of plant steroid hormone (brassinosteroid) signaling pathways, the TNT^® T7 Coupled Reticulocyte Lysate System was used for in vitro transcription/translation of proteins that were then used in GST pull-down assays to study protein:protein interactions. (2429)

Notes: Promega's Anti-Cytochrome C monoclonal antibody was used  in immunocytochemistry to detect mitochondrial release of Cytochrome C in apoptotic IMR90-E1A cells microinjected with caspase-2 expressing constructs. For this procedure, cells were fixed in 3% paraformaldehyde, washed, then permeabilized with 0.1% Triton X-100. Dilutions of Anti-Cytochrome C monoclonal antibody were applied, then visualized using Alexa Fluor 633 (Molecular Probes)-labeled secondary antibody, and a Leica TCS NT laser scan microscope equipped with a 488–534nm argon laser and a 633 nm He-Ne laser. The researchers also used the TNT^® T7 Coupled Reticulocyte Lysate System for in vitro transcription/translation of RAIDD/CRADD and caspase-2 deletion mutants. Templates for the transcription/translation reaction consisted of cDNA cloned into pCDNA₃-HA vectors.  The proteins of interest were labeled with ³⁵S-labeled amino acids and were used in protein association assays with GST-caspase-2 protein.  (2667)

Notes: T4 Polynucleotide Kinase was used to phosphorylate primers before ligating them into a phagemid vector to allow cloning of Sfi I-restricted DNA fragments. Phagemid was prepared from XL1-Blue cells using the Wizard® Minipreps DNA Purification System. Inserts were PCR amplified from the phagemid and purified using the Wizard® PCR Preps DNA Purification System. In separate experiments, the cDNA for the melanin concentrating hormone receptor 1 cDNA was cloned from total melanocyte RNA using MMLV Reverse Transcriptase. In vitro translation of the cDNA was performed using the TnT T7 Coupled Reticulocyte Lysate System and Canine Pancreatic Microsomal Membranes. (2602)

Notes: The authors used Promega's TNT transcription/translation systems to produce ³⁵S labeled substrates for in vitro caspase cleavage assays. (2612)

Notes: In this study, mutations in the CTCF zinc finger transcription factor were found by screening tumor samples from cancer patients. These same mutations were introduced into a cloned version of the gene and the encoded proteins were translated in vitro using the TNT^® T7 Coupled Rabbit Reticulocyte Lysate System.  The translated proteins were used in gel shift assays with radiolabeled CTCF target DNA. The gel-shift binding buffer was supplemented with 0.1mM zinc sulfate. (3133)

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 SV Total RNA Isolation System was used to isolated RNA from the syncytiotrophoblast of human term placentas. The isolated RNA was used for RT-PCR with M-MLV Reverse Transcriptase used for the RT reaction. The 110 kDa PKD2 protein was expressed in vitro with the TNT^® T7 Coupled Reticulocyte Lysate System with and without Canine Pancreatic Microsomal Membranes. The channel activity of the expressed protein was examined and the functional channel could be inhibited by known inhibitors. Expressing the luciferase control with the membranes did not produce the same effect. (2187)

Notes: The murine metalloprotease decysin, which binds zinc, was inserted in a vector containing a carboxyl-terminal myc epitope and a 6X histidine tag and translated using the TNT^® T7 Coupled Reticulocyte Lysate System. The His-tagged protein was purified using a Ni²⁺-NTA column and then tested for processing by the endoprotease furin. (3109)

Notes: The TNT^® T7 Coupled Reticulocyte Lysate System was used to in vitro-label and express the novel 116KDa human Nesprin-1 (nuclear envelope spectrin repeat) protein. The protein was labeled with ³⁵S-methionine in the presence or absence of canine pancreatic microsomes for 2 hours. The researchers also studied whether the expressed protein was inserted into the microsomes. The expressed protein was analyzed after detergent and/or proteinase K digestion of the membranes.  (3018)

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: PCR product was used as a template in an in vitro transcription-translation reaction using the TNT^® T7 Coupled Reticulocyte Lysate System for a protein truncation test (PTT) assay. (1410)

Notes: Genomic DNA was isolated from blood of patients and immediate family members. Exons 32 and 33 of the PLEC1 gene were amplified and tested in the Protein Truncation Test (PTT). Exon 32 was divided into two sections of 1623bp and 2097bp, and Exon 33 was divided into three sections of 2001bp, 2080bp and 2516bp. The TNT^® Coupled Reticulocyte Lysate System was used to express the amplified products. (0459)

Notes: The merlin pcDNA3 plasmids were used as template for a TNT^® T7 Coupled Reticulocyte Lysate System in the presence of 35S-methionine to produce radiolabeled protein for an in vitro protein binding assay. (0337)

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: Reverse transcription reaction was performed in the presence of 25ug/ul Single-Stranded DNA Binding Protein and 1 unit/ul RNasin^® Ribonuclease Inhibitor. PCR product and ³⁵S-methionine were added to the TNT^® Coupled Reticulocyte Lysate System to produce labeled protein in a PTT assay. (1185)

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: The Serine-Threonine Phosphatase Assay System was used to monitor dephosphorylation of two phosphopeptides derived from the SCR homeodomain. Dephosphorylation was performed using the catalytic subunit of the PP2A protein. Authors also used pSI for cloning, protein kinase A enzyme, PKA and PKC assays using biotinylated peptides (SignaTECT® Protein Kinase assay systems), anti-β-galactosidase for Drosophila embryo staining, and TNT® Quick Coupled Transcription/Translation systems to produce mutant and wild type protein for gel shifts. (2150)

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 TNT^® Coupled Reticulocyte Lysate System and the Transcend™ Chemiluminescent Non-Radioactive Translation Detection System were used to produce and label wildtype and mutant human vitamin D receptors. The proteins produced were used for vitamin D binding assays. (0133)

Notes: PCR products were used in TNT^® T7 Coupled Reticulocyte Lysate System. (1215)

Notes: The cDNA for ApoM was translated with the TNT^® Coupled Reticulocyte Lysate System in the presence or absence of the Canine Pancreatic Microsomal Membranes (CMMs). The protein had a higher apparent molecular weight in the presence of the CMMs and the molecular weight was returned to that in the absence of CMMs by treatment with PNGase. (0125)

Notes: The authors identified c-E10, a protein that has an N-terminal caspase-recruiting domain, in a screen for molecules involved in regulation of death receptor signaling pathways. A cDNA encoding viral-E10 was transcribed and translated in vitro using Promega TNT Coupled Reticulocyte Lysate Systems. Additionally, HeLa cells were transfected with a variety of cDNAs together with a β-galactosidase reporter gene, treated with TNFα, and then assayed for cell death using the CellTiter^® Non-Radioactive Cell Proliferation Assay. Expression of c-E10 did not affect the ability of TNFα to induce apoptosis in the transfected cells.  The authors also used the Luciferase Assay System to assess NF-κB activation in the transfected cells. (2502)

Notes: Wildtype and mutant Ca2+-ATPase cDNAs were cloned into the pSP64 Poly(A) Vector and expressed in the TNT® Reticulocyte Lysate System in the presence of the Canine Pancreatic Microsomal Membranes. The membranes were spun out of the reaction and analyzed by SDS-PAGE. Both wildtype and mutant proteins localized to the membranes. (1250)