Notes: Single-stranded 3´-end biotinylated oligonucleotides were captured on the Streptavidin MagneSphere^® Paramagnetic Particles (SA-PMPs) and incubated with heparin-agarose affinity-purified nuclear proteins. The reaction was performed under the conditions used for electrophoretic gel shifts. Proteins were eluted in 1X nonreducing SDS-PAGE sample buffer at 65°C for 3 minutes and analyzed by Southwestern blotting with ³²P-labeled oligonucleotides. The ssDNA-affinity capture worked with the oligonucleotides prebound to the particles or after allowing the interactions to occur in solution prior to capture. The pCI Mammalian Expression Vector was used to express the proteins Purα (321 amino acids) and Purβ (324 amino acids) in mouse embryo-derived AKR-2B fibroblasts. Cell extracts were prepared and assayed by Southwestern blotting. The same proteins were in vitro translated with the TNT^® Coupled Reticulocyte Lysate System and assayed as well. (0929)

Notes: This methods article describes the in vitro expression cloning technique (IVEC) with detailed methods and materials descriptions. Plasmid or phage cDNA libraries were constructed in appropriate T7, T3 or SP expression vectors, and DH10B E. coli were transformed with the libraries. Plasmid minipreps were performed on pools of bacterial colonies, and the pooled cDNAs were used as templates in TNT^® Coupled Reticulocyte Lysate System reactions. Proteins were radioactively labeled, and proteins that were phosphorylated or degraded during mitosis were identified by incubation of radiolabeled library pools with mitotic or interphase Xenopus extracts. Proteins were also tested as putative apoptotic caspase substrates using apoptotic and control Jurkat lymphoma cell extracts. In addition, unlabeled proteins were tested for DNA-binding activity in EMSAs. (2049)

Notes: The CellTiter 96^® AQ_ueous Non-Radioactive Cell Proliferation Assay (MTS/PMS) was used to measure the proliferation of S91 cells. The TNT^® Coupled Reticulocyte Lysate System was used to translate the retinoic acid receptor and the retinoid X receptor. These were then assayed in gel shifts. The recombinant human AP-1 and the AP-1 Consensus Oligonucleotides were used in the gel shift assays as well. (0360)

Notes: This methods article describes the in vitro expression cloning technique (IVEC) with detailed methods and materials descriptions. Plasmid or phage cDNA libraries were constructed and transformed into the appropriate bacterial strains and pooled constructs were then used as templates in TNT^® Coupled Reticulocyte Lysate System reactions. Proteins were radioactively labeled, and proteins that were phosphorylated or degraded during mitosis were identified by incubation of radiolabeled library pools with mitotic or interphase Xenopus extracts. Proteins were also tested as putative apoptotic caspase substrates using apoptotic and control Jurkat lymphoma cell extracts. (0305)

Notes: The rat β1b and β4 as well as the rabbit β2a and β3 were in vitro transcribed/translated in the presence of [³⁵S]methionine in the TNT^® Coupled Reticulocyte Lysate System. The labeled proteins were analyzed for binding to an α1c subunit-GST fusion protein. (0832)

Notes: Luciferase studies were performed in SV40 T type II cells and IMR90 fibroblasts using constructs prepared in the pGL3 Basic Vector. Luciferase activity was measured with the Luciferase Assay System. The hepatocyte nuclear factor 3beta was produced in vitro with the TNT^® Coupled Reticulocyte Lysate System and used for gel shift analysis. (0495)

Notes: Full-length ER was expressed using the TNT® T7 Reticulocyte Lysate System. EMSAs were performed with modifications to reflect dependence on ligand. Binding reactions contained ER in reticulocyte lysate, unlabeled non-specific DNA and either hormone, various concentrations of tests compounds or ethanol and then labeled, specific DNA (30,000cpm) was added. (Parallel series was run with E2 to determine maximal binding.) The assay accurately distinguishes between compounds that do or do not act as ligands, and it allowed relative binding affinity values to be assigned for the test compound. (1842)

Notes: Estrogen receptor (ER) was expressed in the TNT^® T7 Coupled Reticulocyte Lysate System. EMSAs were performed to test whether ER can bind to corticotropin-releasing factor (CRF) promoter half-sites. The in vitro translated ER was tested for binding to CRF promoter half-site oligonucleotides: The data do not support an interaction between ER and the CRF promoter half-sites. (1799)

Notes: Sixty patients were screened for autoantibodies to IA-2 (a protein tyrosine phosphatase-like molecule), IA-2ic (the intracellular fragment of IA-2) and GAD65 (glutamic acid decarboxylase) using RIAs. Full-length human cDNAs were cloned into the pGEM^®-4Z Vector and expressed in the TNT^® Coupled Reticulocyte Lysate System in the presence of [³⁵S]methionine. Incorporated radiolabel was determined by precipitation and scintillation counting. Immunoprecipitation was performed with 5µl of serum and counted on a multiwell counter. Antibodies to IA-2 or GAD65 were detected in 87% of newly diagnosed type 1 diabetic patients and in all 56 prediabetic samples from 11 twins. The frequencies of antibodies to IA-2ic were similar in type 1 diabetics. (1763)

Notes: The system was used to isolate poly A+ RNA from the total RNA of 12 different human cell lines using the PolyATtract^® mRNA Isolation System. The isolate RNA was used for Northern analysis. The fmol^® DNA Cycle Sequencing System and TNT^® Coupled Reticulocyte Lysate System were also used in the study. (1651)

Notes: Both ZEB and the DNA-binding domain of ZEB were subcloned into Promega's pCI-neo Mammalian Expression Vector just behind a FLAG epitope. After cotransfection with reporter vectors, the effect of the expressed protein was assessed in 10T1/2 fibroblasts and C2C12 cells. The two protein inserts were subcloned into a T3 RNA polymerase binding site bearing plasmid and the flag-tagged proteins were expressed in vitro with the TNT^® Coupled Reticulocyte Lysate System. (0542)

Notes: The authors compared translation of proteins with and without an RBS-hybrid-spacer from Ner. Transcripts that contained the RBS-hybrid-spacer were robustly translated, whereas transcripts with the LacZ RBS spacer were inefficiently translated. Translation was performed using the TNT^® T7 Coupled Reticulocyte Lysate System (2219)

Notes: Human PPAR-gamma and retinoid-x-receptor (RXRalpha) were expressed in the TNT® Coupled Reticulocyte Lysate System. The proteins were assessed for binding to double-stranded oligonucleotides with repeats of the sequence TGACCT, separated by 0-5 nucleotides or the ACO sequence by EMSAs. The hPPARalpha recognizes several direct repeat elements, and bound to the different oligonucleotides in a heterodimer with RXRalpha. (1806)

Notes: A novel mouse gene showing high homology to the MAP kinase phosphatase gene family was cloned, and the cDNA was transcribed and translated using the TNT® SP6 Coupled Reticulocyte Lysate System with [35S]methionine. The gene produced a protein of approximately the predicted size, but the protein had slightly aberrant migration of the protein, possibly due to polyglycine or polyserine stretches and/or the proline-content of the protein. (0258)

Notes: Both the b and b' meprin isoforms were synthesized in the presence or absence of the microsomes. The ~70kDa protein was shifted to ~110kDa in the presence of the membranes. (2046)

Notes: Recombinant Gs and adenylyl cyclase (ACIV) proteins were produced using the TNT^® T7 Coupled Reticulocyte Lysate System. The Gs alpha proteins were altered in their N-termini and shown to have altered activity. Both Gs alpha52 and Gs alpha54 can form a heterotrimer with G beta gamma and could activate adenylylcyclase. When incorporated into membranes, Gs alpha54 retained these abilities. (0168)

Notes: CREB was transcribed and translated in the TNT® T7/SP6 Coupled Reticulocyte Lysate System in the presence of [35S]methionine. CREB protein was tested for binding to a consensus CRE. DNA-CREB complexes were formed corresponding to homodimers of the I-CREB(l) and I-CREB(s) as well as heterodimers among the two I-CREB, full-length activator CREBs and truncated CREB. (1820)

Notes: The catalytic subunit from the Na^+_-K⁺-ATPase was expressed in Wheat Germ Extracts or Rabbit Reticulocyte Lysates. In immunoblots using an antibody that is specific to the first 9 residues of the predicted protein, the in vitro synthesized protein but not the in vivo synthesized protein was detected, suggesting that the protein is processed in vivo. Processing was inefficient or absent in the TNT^® Coupled Reticulocyte Lysate Systems. (0546)

Notes: DNA binding was examined using EMSAs. Proteins encoded by CREB, CREB-W and CREB-WZ were expressed in TNT® T7 or SP6 Reticulocyte Lysate Systems. Extracts were incubated with a 32P-labeled CRE-containing oligo. CREB produced a single complex with the CRE probe, and CREB-W and CREB-WZ produced several smaller complexes. (1801)

Notes: Wild type and mutant versions of the catalytic subunit (C) of bovine protein kinase A were expressed in the TNT^® Coupled Reticulocyte Lysate System. Immunoprecipitation of the in vitro translated proteins verified the specificity of alphaP-2 antibodies. (1762)

Notes: Eighteen sporadic neoplasms of ampullary origin were examined for the involvement of mutations in APC, Ras, p53 and chromosomal 5q21 allelic losses. 5q21 loss (including APC) occurred in 50% and APC mutations occurred in 17% of the ampullary tumors. 5q21 loss and mutations in APC and Ras occur in early tumor development, and p53 inactivation is associated with progression of malignancy. The researchers used PTT for APC mutation detection in the TNT® T7 Coupled Reticulocyte Lysate System with chemiluminescent labeling instead of radiolabeling of proteins. (2056)

Notes: The full-length and extracellular region of the human TSH-R (TSH-R and TSH-R.E, respectively) were expressed in the TNT^® Coupled Reticulocyte Lysate System in the presence of [³⁵S]methionine. First, immunoprecipitation was performed to the proteins with one rabbit polyclonal antiserum and three murine antibodies that recognize 3 different determinants on the proteins. From the full-length receptor, the antibodies recognize the 97kDa as well as other smaller bands. The TSH-R was also immunoprecipitated with sera from Graves’ Disease patients. The majority of sera precipitated the TSH-R 97kDa and TSH-R.E 50kDa proteins, with some sera precipitating other, smaller proteins. There was variability in strength between different samples. (1773)

Notes: PTT was used to rapidly screen the BRCA1 gene. Transcription and translation was performed on five overlapping fragments (amplified by PCR) of the entire coding region of the BRCA1 cDNA or genomic DNA using the TNT^® T7 Coupled Reticulocyte Lysate System. (1144)

Notes: The protein truncation test was performed on PCR products from the BRCA2 gene. The PCR products were used in the TNT^® T7 Coupled Reticulocyte Lysate System to express the protein, and the protein was analyzed by SDS-PAGE. (0834)

Notes: The E11 cDNA was transcribed and translated in vitro using the TNT^® T3 Coupled Reticulocyte Lysate System in the presence of [³⁵S]methionine. The protein was immunoprecipitated with mAb E11 and with a control IgG1 antibody. When the membrane was reacted with the mAb E11, the appropriate molecular weight bands were seen, verifying that the cDNA codes for E11. (1778)