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FEBS J. 280, 1773-81. A convenient luminescence assay of ferroportin internalization to study its interaction with hepcidin. 2013

Song, G., Jiang, Q., Xu, T., Liu, Y.L., Xu, Z.G., and Guo, Z.Y.

Notes: Hepcidin is a small peptide secreted by the liver that plays a key role in iron homeostasis by binding and internalizing the iron efflux transporter ferroportin (Fpn). The authors of this paper used NanoLuc® luciferase-tagged and GFP-tagged Hepcidin fusion proteins to measure the internalization of Fpn in HEK293T cells. Once the NanoLuc®-tagged Fpn was internalized, luminescence was significantly decreased. The authors coexpressed both NanoLuc®-tagged Fpn and GFP-tagged Fpn using a doxycycline-inducible bidirectional promoter and were able to measure hepcidin-induced Fpn internalization qualitatively (GFP fluorescence) and quantitatively (bioluminescence).  (4436)

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J. Cell Biol. 202, 579–95. A new method for high-resolution imaging of Ku foci to decipher mechanisms of DNA double-strand break repair. 2013

Britton, S. Coates, J. and Jackson, S.P.

Notes: The authors transiently transfected 5 × 106 HEK293-AAV cells per 100cm dish using the FuGene® HD Transfection Reagent, 9µg of DNA and a reagent:DNA ratio of 3:1. (4431)

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Biochimie Sept 18, Epub ahead of print. doi: 10.1016/j.biochi.2013.09.008.. A novel untrasensitive bioluminescent receptor-binding assay of INSL3 through chemical conjugation with nanoluciferase. 2013

Zhang, L., Song, G., Xu, T., Wu, Q.P., Shao, X.X., Liu, Y.L., Xu, Z.G., and Guo, Z.Y.

Notes: These authors developed a sensitive receptor-binding assay for detection of interactions between the peptide hormone Insulin-like peptide-3 (INSL3) and the relaxin family peptide receptor RXFP2.  Recombinant INSL3 was tagged with NanoLuc® Luciferase by chemical modification of INSL3 to include an active disufide bond, and engineering of a 6× His-Cys-NanoLuc with an exposed N-terminal cysteine. The NanoLuc®-conjugated INSL3 was used to monitor the receptor-binding of a variety of ligands. (4438)

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J. Virol. 87(21), 11955-11962. Assessing activity and inhibition of Middle East respiratory syndrome coronavirus papain-like and 3C-like proteases using luciferase-based biosensors. 2013

Kilianski, A. Mielech, A.M., Deng, X., and Baker, S.C.

Notes: These authors generated luciferase biosensors and used them to screen for potential inhibitors of Middle East respiratory syndrome coronavirus (MERS-CoV) replication. The biosensors were developed using the pGloSensor™-30F plasmid backbone and were engineered to include cleavage sites for the viral papain-like protease (PLpro) or 3-chymotrypsin-like protease (3CLpro). Lytic, endpoint assays and live-cell assays were performed to detect protease activity. The authors identified a small-molecule inhibitor that blocked the activity of MERS-CoV 3CLpro. These biosensor-based assays allowed rapid evaluation of viral protease activity and screening for protease inhibitors. (4526)

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Proc. Natl. Acad. Sci. USA 110, 9577–9582. Blast resistance of CC-NB-LRR protein Pb1 is mediated by WRKY45 through protein-protein interaction. 2013

Inoue, H., Hayashi, N., Matsushita, A., Xinqiong, L., Nakayama, A., Sugano, S., Jiang, C.J. and Takatsuji, H.

Notes: To understand the mechanism of Panicle blast 1 (Pb1) gene-mediated resistance to rice blast, a rice fungal disease, researchers investigated Pb1 interacted with a transcription factor involved in resistance, WRKY45 that is regulated by the ubiquitin system. To study how these proteins interacted, inner rice leaf sheaths were bombarded with gold particles coated with 0.5 µg of effector plasmid, 0.3 µg of NanoLuc® luciferase reporter and 0.1 µg of reference Renilla luciferase. After incubating overnight at 28°C, samples were ground in liquid nitrogen and reporter activities assayed using the Dual-Glo® Luciferase Reporter Assay System and Nano-Glo® Luciferase Assay System. The Renilla luciferase gene was also split into an N-terminal construct and C-terminal construct, expressed in rice protoplasts and assayed for reconstituted Renilla luciferase activity. Expression was normalized to firefly luciferase. (4510)

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BMC Ecol. 13. Capturing chloroplast variation for molecular ecology studies: A simple next generation sequencing approach applied to a rainforest tree 2013

McPherson, H., van der Merwe, M., Delaney, S.K., Edwards, M.A., Henry, R.J., McInstosh, E., Rymer, P.D., Milner, M.L., Siow, J. and Rossetto, M.

Notes: The authors of this study used next generation sequencing technology to assemble chloroplast genome and detect single nucleotide polymorphisms for a rainforest tree species. Genomic DNA was extracted from leaf samples, and 2-3 extractions per individual tree were pooled. Genomic DNA was quantitated using the QuantiFluor® dsDNA System on the SpectraMAX Gemini XPS detector prior to next generation sequencing. (4509)

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J. Biol. Chem. 288, 34470-83. Chemotype-selective modes of action of κ-opioid receptor agonists. 2013

Vardy E., Mosier P.D.,  Frankowski K.J., Wu H., Katritch, V., Westkaemper R.B., Aubé, J., Stevens R.C., Roth B.L.

Notes: The authors sought to determine whether different residues had specific roles in binding and activation of the k-opioid receptor (KOR) by agonist molecules with distinct chemotypes. For function assays they introduced point mutations into human KOR using site-directed mutagenesis. All mutations were verified by Sanger automated sequencing. HEK293T cells were cotransfected with pGloSensor™-22F cAMP Plasmid plus the different KOR variants. Cells were plated, and after 24 hours, medium replaced with drug buffer and the different drug treatments. cAMP production was detected by treatment with isoproterenol in GloSensor™ Reagent. (4519)

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J. Bacteriol. 195, 708–16. Chlamydia trachomatis Tarp harbors distinct G and F actin binding domains that bundle actin filaments. 2013

Jiwani, S. et al.

Notes: The authors transiently transfected 2 × 105 HeLa cells grown on coverslips in a 6-well plate using the FuGene® HD Transfection Reagent, 2.5µg of DNA and a reagent:DNA ratio of 8:2.5. (4433)

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Int. J. Syst. Evol. Microbiol. 63, 1062–7. Chryseobacterium rigui sp. nov., isolated from an estuarine wetland. 2013

Park, S.-J., Choi, J.-H. and Cha, C.-J.

Notes: In this study the Wizard® Genomic DNA Purification Kit was used to isolate DNA from Chryseobacterium rigui. (4318)

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ACS Chemical Biology 8, 1018–26. Conformation guides molecular efficacy in docking screens of activated β-2 adrenergic G protein coupled receptor. 2013

Weiss, D.R. et al.

Notes: The authors investigated the utility of virtual screening to identify agonists for the active form of the G protein-coupled β2 adrenergic receptor (β2AR). During screening, they maintained the active form of β2AR with conformationally selective antibodies and used cAMP levels to quantify β2AR activity. cAMP levels were determined using the GloSensor™ cAMP biosensor, a genetically modified form of firefly luciferase into which a cAMP-binding protein moiety is inserted such that cAMP binding induces a conformational change and leads to increased light output. (4517)

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Veterinary Microbiology 162, 858–65. Construction and evaluation of an Edwardsiella ictaluri fhuC mutant. 2013

Abdelhamed, H. et al.

Notes: In this study the Wizard® Genomic DNA Purification Kit was used to isolate DNA from Edwardsiella ictaluri fhuC. (4328)

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Methods 6, 283–291. Construction of Specific Parallel Amplification of RNA Ends (SPARE) libraries for the systematic identification of plant microRNA processing intermediates. 2013

Schapire, A.L., Bologna, N.G., Moro, B., Zhai, J., Meyers, B.C. and Palatnik, J.F.

Notes: The authors developed a complete method for making cDNA libraries containing miRNA processing-intermediates that used the RQ1 RNase-Free DNase and Wizard® SV Gel and PCR Clean-Up System. (4920)

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Soil Biol. Biochem. 64, 18–27. Contrasting Euryarchaeota communities between upland and paddy soils exhibited similar pH-impacted biogeographic patterns 2013

Wu, H-W., Zhang, L-M, Yuan, C-L. and He, J-Z.

Notes: Products from PCR of the archaeal 16SrRNA gene were purified using the Wizard® SV Gel and PCR Clean-Up Kit before pyrosequencing. (4549)

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N. Engl. J. Med. 369(2), 164-71. Delayed puberty and estrogen resistance in a woman with estrogen receptor α variant. 2013

Quaynor, S.D., Stradtman, E.W. Jr., Kim, H.G., Shen, Y., Chorich, L.P., Schreihofer, D.A., and Layman, L.C.

Notes: In this study, FuGENE® 6 was used to perform transient transfection of COS-7 cells. Plasmids containing mutated or non-mutated copies of the estrogen receptor gene ESR1 were transfected together with luciferase reporter constructs containing estrogen response elements upstream of the luciferase gene. Transactivation of the estrogen response element was reduced in the mutated estrogen receptor compared with the nonmutated  receptor. (4407)

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Folia Microbiol. 58, 623–30. Detection and quantification of probiotic strain Lactobacillus gasseri K7 in faecal samples by targeting bacteriocin genes. 2013

Treven, P., Turkova, K., Trmčić, A., Obermajer, T., Rogelj, I. and Matijašić, B.B.

Notes: The authors were interested in quantitating the presence as well as the prevalence of Lactobacillus gasseri K7 in humans that did not consume the probiotic bacteria. Fecal samples from 45 healthy adults were collected, frozen, diluted, centrifuged and digested with proteases. After sonication, DNA was extracted using the Maxwell® 16 Tissue DNA Purification kit on the Maxwell® 16 instrument. This same kit and instrument also were used to isolate bacterial DNA from 1ml bacterial cultures. The purified DNA was PCR amplified using primers for gassericin K7 A and K7 B (bacteriocin) genes and GoTaq® Flexi DNA Polymerase in Green GoTaq® Flexi Buffer for 30 cycles. PCR products were analyzed by 1.8 % agarose gel electrophoresis. (4522)

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Forensic Sci. Int. Genet. 7, 129–35. Developmental validation of the PowerPlex® 18D System, a rapid STR multiplex for analysis of reference samples. 2013

Oostdik, K., French, J., Yet, D., Smalling, B., Nolde, C., Vallone, P.M., Butts, E.L., Hill, C.R., Kline, M.C., Rinta, T., Gerow, A.M., Allen, S.R., Huber, C.K., Teske, J., Krenke, B., Ensenberger, M., Fulmer, P. and Sprecher, C.

Notes:   (5200)

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Forensic Sci. Int. Genet. 7, 240–50. Developmental validation of the PowerPlex® Y23 System: A single multiplex Y-STR analysis system for casework and database samples 2013

Thompson, J.M., Ewing, M.M., Frank, W.E., Pogemiller, J.J., Nolde, C.A., Koehler, D.J., Shaffer, A.M., Rabbach, D.R., Fulmer, P.M., Sprecher, C.J. and Storts, D.R.

Notes:   (5203)

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J. Clin. Microbiol. 51, 745–51. Efficient depletion of host DNA contamination in malaria clinical sequencing. 2013

Oyola, S.O. et al.

Notes: Human Genomic DNA: Male was used to construct a reference mock clinical sample. (4545)

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Epigenetics 8, 534–541. First evidence of DNA methylation in insect Tribolium castaneum: Environmental regulation of DNA methylation with heterochromatin 2013

Feliciello, I., Parazajder, J., Akrap, I. and Ugarović, Ð.

Notes: GoTaq® Green Master Mix was used to amplify Tribolium castaneum  satellite DNA that had been bisulfite treated to detect methylated cytosines. The bisulfite-treated satellite DNA was amplified using methyl-specific primers in a total volume of 30µl using 2X GoTaq® Green Master Mix, 2mM mix of the primer sets, and 1µl of the bisulfite modified DNA.

  (4356)

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PLos ONE 8, e67151. First transcriptome and digital gene expression analysis in Neuroptera with an emphasis on chemoreception genes in Chrysopa pallens (Rambur) 2013

Li, Z.Q., Zhang, S., Ma, Y., Luo, J.Y., Wang, C.Y., Lv, L.M., Dong, S.L. and Cui, J.J.

Notes: RNA was isolated from adult C. pallens (insect) tissues using the SV Total RNA Isolation System prior to cDNA library construction and sequencing on the Illumina HiSeq™ platform. To validate NGS sequence alignment, end-to-end PCR was performed. PCR products were purified using the Wizard® SV Gel and PCR Clean-Up System prior to cloning into a T vector. To verify the identified differentially expressed genes, quantitative real-time PCR (RT-qPCR) was used. Total RNA was extracted and cDNAs synthesized using the Reverse Transcription System prior to qPCR. (4560)

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PLos ONE 8, e67151. First Transcriptome and Digital Gene Expression Analysis in Neuroptera with an Emphasis on Chemoreception Genes in Chrysopa pallens (Rambur). 2013

Li, Z.Q., Zhang, S., Ma, Y., Luo, J.Y., Wang, C.Y., Lv, L.M., Dong, S.L. and Cui, J.J.

Notes: The authors isolated RNA from adult C. pallens (insect) tissues using the SV Total RNA Isolation System prior to cDNA library construction and NGS at BGI using Illumina Instruments. (4915)

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Proc. Natl. Acad. Sci. USA 110, 18620–5. GATA-1 regulates the generation and function of basophils. 2013

Nei, Y., Obata-Ninomiya, K., Tsutsui, H., Ishiwata, K., Miyasaka, M., Matsumoto, K., Nakae, S., Kanuka, H., Inase, N. and Karasuyama, H.

Notes: RNA was isolated from mouse basophils following fluorescence-activated cell sorting and used in RT-qPCR analysis. (4443)

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PLos ONE 8, e84071. Gene expression profiling of early hepatic stellate cell activation reveals a role for Igfbp3 in cell migration. 2013

Mannaerts, I., Schroyen, B., Verhulst, S., Van Lommel, L., Schuit, F., Nyssen, M. and van Grunsven, L.A.

Notes: Mouse hepatic stellate cells (HSCs) were cultured with valproic acid for various times. Total RNA was isolated and analyzed for multiple transcripts by RT-qPCR using the dye-based GoTaq® RT-qPCR System. Total RNA was isolated with the ReliaPrep™ RNA Cell Miniprep System and used for RT-qPCR and expression profiling with an Affymetrix genechip array. (4610)

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PLos ONE 8, e62137. Genotyping-by-Sequencing (GBS): A novel, efficient and cost-effective genotyping method for cattle using next-generation sequencing 2013

De Donato, M., Peters, S.O., Mitchell, S.E., Hussain, T. and Imumorin, I.G.

Notes: The authors of this study sought to apply a genotyping-by-sequencing approach to genotype cattle from the US and Africa. They collected blood samples from 47 unrelated animals slaughtered in the US and Nigeria. Genomic DNA was extracted and then quantitated using the QuantiFluor® dsDNA System on a SpectraFluor Plus plate-format fluorometer before being used for next-generation sequencing. (4508)

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Cell 153, 1327-1339. HIF1A Employs CDK8-Mediator to Stimulate RNAPII Elongation in Response to Hypoxia. 2013

Galbraith, M., Allen, M., Bensard, C., Wang, X., Schwinn, M., Qin, B., Long, H., Daniels, D., Hahn, W., Dowell, R., and Espinosa, J.

Notes: These authors identified a previously unknown interaction between the transcription factor HIF1A and the cyclin-dependent kinase CDK8 (a component of the Mediator complex) in the regulation of genes associated with cellular survival under low-oxygen conditions. As part of the study, HaloTag technology was used to identify proteins interacting with CDK8 in a colorectal cancer cell line. Specifically, cells were transfected with CDK8 and CDK19 HaloTag fusion constructs obtained from Kazusa Institute. The cell lysates were then used in pulldown assays to capture interacting proteins. The results showed that CDK8 and CDK19 are present in mutually exclusive Mediator complexes. Details of the transfection are as follows: HCT116 cells were plated in 150 mm dishes and grown to 70%–80% confluence before transfection with 30 μg of plasmid DNA using FuGENE HD Transfection Reagent. (4355)

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