Notes: Increasing residues of the chloride channel were fused to a prolactin epitope and translated in vitro using Promega's Rabbit Reticulocyte Lysate in the presence of Canine Microsomal Membranes. The proteins were subjected to a proteinase K digest in the presence or absence of Triton X-100. The protease digestions were stopped with PMSF and the translation reaction analyzed by Western blotting. The protease protection assays were used to define the topology of the channel. The chloride channel crosses the membrane at least 10 times. Very good protocol for the protease protection assay. (1596)

Notes: The extracellular domain of the receptor was expressed in vitro using Promega's Rabbit Reticulocyte Lysate in the presence or absence of the microsomes. The 60kDa extracellular domain is processed to a 70kDa glycoprotein in the presence of the membranes. The paper also shows a titration of the membranes in the translation reaction from 1-4µl per reaction. More protein is processed with increasing amounts of the microsomes. (1627)

Notes: Truncation mutants of the protein of interest were translated in the presence of the membranes. The membranes were pelleted and analyzed directly or treated with endoglycosidase F. The supernatents from the centrifugation were acetone precipitated and analyzed as well. (1602)

Notes: The 3AB protein was fused with a FLAG tag. The rabbit lysate was centrifuged prior to use to remove any protein aggregates. The reaction was set up in a 200µl volume with the 3AB protein transcript and a b globin mRNA. The reaction was split into two 100µl reactions and to one 25 equivalents of membranes were added and to the other, just buffer. The translation reactions were allowed to procede for 30min at 30°C, then translation stopped by the addition of cycloheximide to 300µM. The reactions were centrifuged at through a sucrose gradient at 86,000 x g. Fractions were removed and subjected to immunoprecipitation and gel analysis. The 3AB protein was found to be membrane associated. Further studies with the addition of the Canine Microsomal Membranes after translation and cycloheximide demonstrated a membrane association after translation. (1599)

Notes: In this report, the Rabbit Reticulocyte Lysate was used as a model for the eukaryotic cytosol to identify chaperones involved in protein renaturation. Geldanamycin inhibits the rate of luciferase renaturation, indicating that hsp90 is involved in luciferase folding. The chaperone machinery appears to be the same as that for glucocorticoid or progesterone steroid hormone receptors. (0265)

Notes: The novel UT1 urea transporter was translated in vitro in the presence or absence of the microsomes. In the absence of the microsomes, the protein migrates at 76kDa and in the presence, 86kDa. The data is not shown but described in the text. (1597)

Notes: The RSP2 protein was in vitro translated using Promega's Rabbit Reticulocyte Lysate in the presence of microsomes. The protein was not protected from proteinase K digestion and deemed to be a cytoplasmic protein. (1626)

Notes: Native and mutated AR cDNAs were transcribed with the TNT® T7 Coupled Reticulocyte Lysate System. The reaction mixtures were divided and supplemented with either androgen or vehicle. For EMSAs, the preincubated receptors were placed in binding reactions with [32P]-labeled dsDNA corresponding to a glucocorticoid/androgen-responsive element (ARE). All AR receptors with intact DNA-binding domains formed specific complexes with the ARE. The presence of active hormone altered the mobility of receptor-DNA complexes probably due to a conformational change. Mutant proteins could form heterodimers with native AR that interacted with DNA. Androgen receptor (AR) was translated using the Rabbit Reticulocyte Lysate System, and RelA was translated using Wheat Germ Extract. Coimmunoprecipitation was performed with anti-AR or anti-RelA, but no specific association between AR and RelA was detected (even with the cross-linking agent dithiobis(succinimidylpropionate) present). This finding does not exclude the possibility that AR-RelA interactions occur in vivo. (1669)

Notes: In this report, Hsp90, in cooperation with Hsc70, p60 and other factors, was shown to mediate the refolding of thermally denatured proteins, such as firefly luciferase, both in vivo and in vitro. Luciferase-myc-His, generated in E. coli, was placed in desalted Nuclease-Treated Rabbit Reticulocyte Lysate System (RRL), or luciferase was translated in the RRL. Luciferase:chaperone complexes were generated either by adding the luciferase-myc-His to the RRL and treating with apyrase or by incubation of native luciferase in the RRL followed by treatment with apyrase. The complexes were immunoisolated, and the proteins in them were characterized, including Hip, Hsp40 and p23. Incubation of lysates with geldanamycin and herbimycin A indicated that Hsp90 plays a role in ATP-dependent luciferase processing. (0414)

Notes: The protein with six membrane-spanning domains was translated using Rabbit Reticulocyte Lysate in the presence of Canine Microsomal Membranes. The protein was N-glycosylated and this glycosylation was sensitive to endoglycosidase H. Mutants containing one to all six transmembrane domain were constructed and immunoprecipitates tested for susceptibility to protease digestion and determination of membrane topology. The full protein could only be inserted cotranslationally since incubation of translation products with puromycin and the membranes produced no protection from the proteases. (1648)

Notes: RNA coding for a type-III procollagen mini-gene was translated in the Rabbit Reticulocyte Lysate System in the absence of DTT. The polypeptides produced were treated with NEM and immunoprecipitated with antibodies specific for type-III procollagen and separated with or without reduction. Translation products from mutants were digested with chymotrypsin, trypsin and pepsin in the presence and absence of alpha, alpha´-dipyridyl to determine if a correctly aligned triple helix had formed in the mutants. (1761)

Notes: Capped transcripts were prepared with the T7 RNA Polymerase and translated in the presence of the membranes. Prior to analysis by SDS-PAGE, the reactions were layered on 200µl of a sucrose solution and spun at 70,000 rpm. The pellets were solubilized in SDS sample buffer. The expressed protein was treated with endoglycosidase F to demonstrate the presence of glycosylation. (2012)

Notes: The Protein Truncation Test (PTT) is investigated as a rapid, first mutation screening approach in exon 11 of the BRCA1 gene. Eighty-one percent of the mutations found in 63 patients are truncation mutations, and 49% of these mutations are in exon 11 so the PTT is a sound screening method. The Wheat Germ Extract performed better than the Rabbit Reticulocyte Lysates with the primer set used. The investigators suggest that the choice of translation be empirically determined for each primer set used. (0538)

Notes: When Tralpha1 and Tralpha2 are translated in the Rabbit Reticulocyte Lysate System, their binding to the TR element is significantly reduced (not detectable in EMSA, very weak in ABCD assay) due to phosphorylation of the proteins in the Reticulocyte Lysate. Dephosphorylation or serine to alanine substitutions improves DNA binding. (1822)

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)

Notes: A clone of the lysostaphin gene was modified to include eukaryotic gene components and transcribed in vitro. When the RNA was translated in the Rabbit Reticulocyte Lysate, Nuclease Treated System, several products were produced. The lysate had active enzyme that could be translocated (in addition to the preprolysostaphin form), which suggests that staphylococcal mastitis may be controlled by targeting Staphylococcus transcription. (0189)

Notes: Electrophoretic mobility shift assays were carried out with wild type and 870mut androgen receptor (AR) proteins that had been produced using the Rabbit Reticulocyte Lysate System. The mut 807 AR was able to interact in a DNA sequence-specific manner in cell-free conditions. The researchers hypothesize that the 870mut disrupts steroid-induced conformational changes. (2059)

Notes: Enzymatically active diphtheria toxin was formed in vitro by transcription/translation from PCR products carrying the A- and B-fragments of the toxin. T3 RNA Polymerase was used for in vitro transcription and the Rabbit Reticulocyte Lysate System was used for translation of proteins. Both wild type and mutant proteins were produced and assayed. The synthesized proteins were tested for trypsin sensitivity, ability to inhibit protein synthesis, binding and translocation in Vero cells. Considerable alterations can be made to the C-terminal region of the protein without blocking translocation. Some mutant toxins were less toxic than wild type toxins in unnicked form, but when nicked most exhibited the same toxicity. (2226)

Notes: The authors used Promega's Rabbit Reticulocyte Lysate System to express proteins used for gel shift assays (EMSA). (2238)

Notes: Proteins produced by in vitro translation in the Rabbit Reticulocyte Lysate System were used in a gel shift assay. (0131)

Notes: Authors used Rabbit Reticulocyte Lysate to gel shift with TFIID (TATA Binding Protein). (0957)

Notes: Promega's Rabbit Reticulocyte Lysate System was used for in vitro translation of proteins for gel shift assays. (2018)

Notes: Protein products translated in vitro in Rabbit Reticulocyte Lysate and Wheat Germ Extract were used for gel shift assays. (0245)