Notes: Wildtype and albino mutant tyrosinase were translated in Rabbit Reticulocyte Lysate, Nuclease Treated, supplemented with canine microsomal membranes or semipermeabilized melanocytes. After translation, SP-melanocytes were isolated and resuspended in Rabbit Reticulocyte Lysate, Untreated, in the presence of an ATP regenerating system, and ER-associated degradation of the proteins was evaluated. (3375)

Notes: Rabbit Reticulocyte Lysate, Untreated, was used a source of ribosomes for the assay of Shiga-like toxin-1 to depurinate 28S rRNA. No details of how the ribosomes were purified are provided. (0850)

Notes: The authors used Rabbit Reticulocyte Lysate, Untreated, in an in vitro nuclear transport assay with permeabilized mouse A31 cells grown on coverslips. (0308)

Notes: Promega's Untreated Rabbit Reticulocyte Lysate was used to reconstitute nuclear localization signal-dependent nuclear import into mechanically perforated HTC rat hepatoma cells. The import required the lysate, an energy generating system consisting of creatine phosphokinase, creatine phosphate and ATP. The nuclear import of a fluorescein-tagged protein was followed by confocal laser scanning microscopy. (1404)

Notes: Several different gap junction channel subunit isotypes, also known as connexins, were synthesized in vitro in the presence of Canine Microsomal Membranes. Translation reactions were spun through a sucrose cushion to separate the membrane fraction from the supernatant. Connexins that integrated into the microsomes formed homo- and heterooligomeric structures. The assembled proteins in the microsome were fused with a synthetic membrane and single channel conductance was measured and found to be very similar to wild-type membranes. (1618)

Notes: The paper looked at the insertion of a model protein with one, two and four transmembrane segments and different distributions of positively charged residues in the N-terminal tail and the polar loops. The proteins were translated in the presence of the microsomes. Translocation of polypeptides to the lumenal side of the microsomes was assayed by prevention of N-linked glycosylation through competitive inhibition by the addition of a glycosylation acceptor tripeptide but not by a nonacceptor tripeptide and by proteinase K treatment of the microsomes. All techniques are referenced. (2033)

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: 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: 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)