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Biochemistry 36, 1287-1294. Association of domains within the cystic fibrosis transmembrane conductance regulator. 1997

Ostedgaard, L.S., Rich, D.P., DeBerg, L.G., Welsh, M.J.

Notes: Cystic fibrosis transmembrane conductance regulator (CFTR) was expressed in the TNT® Coupled Reticulocyte Lysate System to confirm its molecular weight. Translation was performed with Canine Pancreatic Microsomal Membranes (CMMs) to verify that the protein is integrated into the membrane. Further, the electrophoretic mobility of CFTR suggests that it is glycosylated. (0570)

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Blood 90, 372-381. Calreticulin biosynthesis and processing in human myeloid cells: Demonstration of signal peptide cleavage and N-glycosylation. 1997

Denning, G.M., Leidal, K.G., Holst, V.A., Iyer, S.S., Pearson, D.W., Clark, J.R., Nauseef, W.M. and Clark, R.A.

Notes: PolyA+ RNA from HL-60 cells was in vitro translated using the Rabbit Reticulocyte Lysate System in the presence or absence of Canine Microsomal Membranes, then immunoprecipitated and analyzed by SDS-PAGE and fluorography. The calreticulin protein was presumably glycosylated by the membranes. The same results were obtained with in vitro transcripts of the calreticulin. (1617)

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J. Biol. Chem. 272, 20756-20763. Characterization of point mutations in patients with X-linked ichthyosis. Effects On the structure and function of the steroid sulfatase protein. 1997

Alperin, E. S. and Shapiro, L. J.

Notes: Transcripts were produced using the RiboMAX™ Large Scale RNA Production System and expressed in the Rabbit Reticulocyte Lysate System with or without Canine Pancreatic Microsomal Membranes (CMMs). Proteinase K protection assays were performed with proteins expressed in the presence of the membranes with and without Triton® X-100 solubilization of the membranes. (1509)

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J. Biol. Chem. 272(33), 20756-20763. Characterization of point mutations in patients with X-linked ichthyosis: Effects on the structure and function of the steroid sulfatase protein. 1997

Alperin, E.S. and Shapiro, L.J.

Notes: The ribonuclease inhibitor was used to protect RNA during the reverse transcriptase reaction for RT-PCR. The RNasin® Ribonuclease Inhibitor is also used in the RiboMAX® System for RNA stabilization during in vitro transcription. Transcripts were produce with the RiboMAX® System and expresed in Rabbit Lysate with or without Canine Microsomal Membranes. Proteinase K protection assays are performed with proteins expressed in the presence of the membranes with and without Triton X-100 solubilization of the membranes. The proteinase K digests were stopped by the addition of PMSF and immediately loading the sample into 100°C SDS Sample buffer. (1670)

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J. Biol. Chem. 272, 31738-31746. Cig30, a mouse member of a novel membrane protein gene family, is involved in the recruitment of brown adipose tissue 1997

Tvrdik, P., Asadi, A., Kozak, L.P., Nedergaard, J., Cannon, B., Jacobsson, A.

Notes: The pCI-neo Mammalian Expression Vector was used to construct a chimeric vector with the neo resistance gene downstream of an internal ribosome entry site, so that the neo gene was transcribed with the Cig30 cDNA and both protein controlled by the CMV promoter. The construct was used to make stable transfectants of the HIB-1B hibernoma cells. Cell lines were analyzed by Northern blotting. The protein was also analyzed in vitro with the Rabbit Reticulocyte Lysate and Canine Pancreatic Microsomal Membranes (CMMs). (0246)

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J. Neurosci. 17, 181-189. Conservation of topology, but not conformation, of the proteolipid proteins of the myelin sheath. 1997

Gow A., Gragerov A., Gard A., Colman D.R., Lazzarini R.A.

Notes: All cDNAs to be translated in vitro were subcloned into the pSP64 Poly(A) Vector. The vector was linearized and RNA transcribed from the SP6 promoter with the Riboprobe® in vitro Transcription System. The in vitro transcribed RNA was translated in the Rabbit Reticulocyte Lysate in the presence or absence of Canine Pancreatic Microsomal Membranes (CMMs). N-linked glycosylation sequences were engineered into the proteolipid protein to determine the orientation of the domains of the protein in the membrane. (1114)

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J. Biol. Chem. 272, 30715-30723. Cotranslational membrane insertion of the serine proteinase precursor NS2B-NS3 (Pro) of Dengue virus type 2 is required for efficient in vitro processing and is mediated through the hydrophobic regions of NS2B. 1997

Clum, S., Ebner, K.E. and Padmanabhan, R.

Notes: The NS2B-NS3 protein was translated in the presence of the microsomes. Cotranslational insertion into the membrane was necessary for full processing of the precursor. As more membranes were added to the reaction, more protein was processed. Incubation of the in vitro reaction after the addition of cycloheximide with the microsomal membranes did not result in processed protein. (1650)

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J. Neurosci. 17, 3379-3391. Frequency-dependent inactivation of mammalian A-type K+ channel KV1.4 regulated by Ca2+/calmodulin-dependent protein kinase. 1997

Roeper, J., Lorra, C., Pongs, O.

Notes: Two site-directed mutants of KV1.4 were expressed in the Flexi® Rabbit Reticulocyte Lysate System in the presence of Canine Pancreatic Microsomal Membranes (CMMs). The membranes were gently pelleted, resuspended and the proteins in vitro phosphorylated with Calmodulin-dependent protein kinase II. (0490)

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Cell 88, 757-766. Frxb, an execreted protein expressed in the Spemann Organizer, binds and inhibits Wnt-8. 1997

Wang. S., Krinks, M., Lin, K., Luyten, F.P. and Moos, M., Jr

Notes: To demonstrate an interaction between Frzb and Wnt proteins in vitro, the proteins were cotranslated using Rabbit Reticulocyte Lysate in the presence of Canine Microsomal Membranes then immunoprecipitated with specific antibodies to either one. Control immunoprecipitations were performed with cotranslations with Frzb and the β-lactamase control or Wnt and the β-lactamase control. The procedure for the immunoprecipitation is referenced. (1603)

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J. Cell Biol. 139, 895-905. Functional expression cloning and characterization of SFT, a stimulator of Fe transport. 1997

Gutierrez, J.A., Yu, J., Rivera, S., Wessling-Resnick, M.

Notes: The cRNA for the Stimulator of Fe Transport (SFT) was in vitro translated with Rabbit Reticulocyte Lysate in the presence of Canine Microsomal Membranes. The RRL was used at 70% of total volume and 2 equivalents of the membranes were used. After translation, the reaction was treated with RNase A to reduce background. The SFT protein is predicted to have six transmembrane domains. (1621)

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Am. J. Physiol. 273, F801-F806. Identification of a new gene product (diphor-1) regulated by dietary phosphate. 1997

Custer, M. , Spindler, B. , Verrey, F. , Murer, H. , Biber, J.

Notes: The TNT® Coupled Reticulocyte System and Canine Pancreatic Microsomal Membranes (CMMs) were used to express a clone isolated through a differential display screen. (1291)

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J. Biol. Chem. 272(32), 19697-19707. Identification of membrane insertion sequences of the rabbit gastric cholecystokinin-A receptor by in vitro translation. 1997

Bayle, D., Weeks, D. and Sachs, G.

Notes: The TNT® Coupled Reticulocyte Lysate System was used to characterize the membrane domain of several polytopic proteins. The two systems employed here use the presence or inhibition of glycosylation as an indicator of membrane insertion. The addition of a core glycosylation site resulted in an increase of ~2.5kDa to the relative molecular mass determined by SDS-PAGE. Using fusion vectors HK-M0 and HK-M1, the ability of each transmembrane domain to insert into the membrane was assessed. Of seven putative membrane segments, some had signal anchor properties, one had stop transfer properties and another had no membrane insertion activity. Combining data from the different systems confirmed seven transmembrane segments. (1649)

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EMBO J. 16, 5807-5818. Interaction of MHC class II molecules with the invariant chain: Role of the invariant chain (81-90) region. 1997

Stumptner, P. and Benaroch, P.

Notes: Reactions were performed at 30°C in a 25µl volume with 3 equivalents of membranes. Following translation, the membranes were lysed with detergent and the proteins immunoprecipitated with various antisera. The immunoprecipitated proteins were also subjected to endoglycosidase H digestion. (1598)

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J. Biol. Chem. 272, 18325-18332. Mapping the Ends of Transmembrane Segments in a Polytopic Membrane Protein. Scanning n-glycosylation mutagenesis of extracytosolic loops in the anion exchanger, band 3 1997

Popov, M., Tam, L.Y., Li, J., Reithmeier, R.A.F.

Notes: The authors used Promega's Canine Pancreatic Microsomal Membranes (CMMs) in combination Promega's Rabbit Reticulocyte Lysate to study the effects of various N-linked glycosylation mutations for TM mapping. Researchers included the detergent octaethylene glycol mono n-dodecyl ether from Nikkol in reactions without the CMMs to prevent aggregation of the proteins. (0541)

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J. Cell Biol. 138, 575-588. Molecular characterization of abLIM, a novel actin-binding and double zinc finger protein. 1997

Roof, D.J., Hayes, A., Adamian, M., Chishti, A.H. and Li, T.

Notes: Three isoforms of the actin-binding LIM protein were expressed in vitro using the TNT® Coupled Reticulocyte Lysate System in the presence of Canine Microsomal Membranes. The expressed proteins were used in an actin binding assay. (1631)

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J. Biol. Chem. 272, 30329-30333. Molecular cloning of a new aquaporin from rat pancreas and liver. 1997

Koyama, Y., Yamamoto, T., Kondo, D., Funaki, H., Yaoita, E., Kawasaki ,K., Sato, N., Hatakeyama, K. and Kihara, I.

Notes: The AQP8 protein was expressed in vitro using the TNT® Coupled Reticulocyte Lysate System in the presence of Canine Microsomal Membranes. The protein demonstrated an increase in molecular weight in the presence of the membranes. Promega's pGEM®-T Vector System and Proteinase K were also used. (1624)

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J. Biol. Chem. 272, 23117-23122. Multiple dimeric forms of human CD69 result from differential addition of N-glycans to typical (Asn-X-Ser/Thr) and atypical (Asn-X-Cys) glycosylation motifs. 1997

Vance, B.A., Wu, W., Ribaudo, R.K., Segal, D.M. and Kearse, K.P.

Notes: The CD69 transcripts were prepared with the T3 RiboMAX® System and translated with the Flexi® System in the presence of the membranes. After 60 minutes at 30°C, the translation reaction was layered onto a sucrose solution and centrifuged at 100,000 x g for 20min. The supernatant was discarded and the pellet was resuspended in SDS-PAGE sample buffer or digested with endoglycosidase H. (1601)

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Biochemistry 36, 11437-11443. Role of ribosomes in reinitiation of membrane insertion of internal transmembrane segments in a polytopic membrane protein. 1997

Wang, C., Chen, M., Han, E., Zhang, J. T.

Notes: The authors used the Rabbit Reticulocyte Lysate together with Canine Pancreatic Microsomal Membranes (CMMs) to study the insertion of internal transmembrane domains. They removed ribosomes from Rabbit Reticulocyte Lysate by centrifugation and replaced with ribosomes isolated from a Wheat Germ Extract and restored functional translation with resulting Rabbit Reticulocyte Lysate supernatant (ribosome-free) fraction. (0196)

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J. Biol. Chem. 272, 13750-13757. The N-terminal domain of human GABA Receptor rho 1 subunits contains signals for homooligomeric and heterooligomeric interaction. 1997

Hackan, A.S., Wang, T.L., Guggino, W.B. and Cutting, G.R.

Notes: The proteins r1 and r2 as well as the N-terminal half of r1 and the C-terminus of r1 were expressed using the Flexi® Rabbit Reticulocyte Lysate System in the presence of Canine Microsomal Membranes. The r1 protein has four membrane spanning domains. The orientation of the protein was determined by proteinase K digestions of intact membranes. Digestions were terminated by the addition of PMSF rather than direct lysis in SDS sample buffer. (1622)

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Proc. Natl. Acad. Sci. USA 94, 7633-7638. Transmembrane topology of a CLC chloride channel. 1997

Schmidt-Rose, T. and Jentsch, T.J.

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)

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J. Biol. Chem. 271(4), 2271-2278. A novel meprin b' mRNA in mouse embryonal and human colon carcinoma cells. 1996

Dietrich, J.M., Jiang, W. and Bond, J.S.

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)

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J. Clin. Invest. 97, 910-914. Autoantibodies to the extracellular domain of the calcium sensing receptor in patients with acquired hypoparathyroidism. 1996

Li.Y., Song, Y.H., Rais, N., Connor, E., Schatz, D., Muir, A. and Maclaren, N.

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)

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J. Biol. Chem. 271, 24817-24843. Cloning and characterization of islet cell antigen-related protein-tyrosine phosphatase (PTP), a novel receptor-like PTP and autoantigen in insulin-dependent diabetes. 1996

Cui, L., Yu, W.P., DeAizpurua, H.J., Schmidli, R.S. and Pallen, C.J.

Notes: The IAR PTP protein (1015 residues) and the B11 protein (820 residues) were synthesized in the presence or absence of the Canine Microsomal Membranes. The predicted protein sequences contain one transmembrane domain. Proteinase K digestion in the presence or absence of Triton X-100 demonstrated the protection of a fragment consistent with the single membrane spanning domain. (1616)

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J. Biol. Chem. 271, 15187-15193. Investigation of myotonic dystrophy kinase isoform translocation and membrane association. 1996

Waring , J.D., Haq, R., Tamai, K., Sabourin, L.A., Ikeda, J.-E. and Korneluk, R.G.

Notes: The MyoD kinase was translated using the TNT® Coupled Reticulocyte Lysate System in the presence or absence of the microsomes. No change in mobility was noted for the MyoD kinase but the a-factor control did get glycosylated. The MyoD kinase did not gain protection from proteinase K but the a-factor was fully protected. (1604)

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J. Biol. Chem. 271(47), 29928-29936. The Dri 42 gene, whose expression is up-regulated during epithelial differentiation, encodes a novel endoplasmic reticulum residuent transmembrane protein. 1996

Barilá, D., Plateroti, M., Nobili, F., Muda, A.O., Xie, Y., Morimoto, T. and Perozzi, G.

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)

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