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Clin. Vaccine Immunol. 13, 930–935. DNA vaccine using Mycobacterium bovis Ag85B antigen induces partial protection against experimental infection in BALB/c mice. 2006

Teixeira, F.M., Teixeira, H.C., Ferreira, A.P., Rodrigues, M.F., Azevedo, V., Macedo, G.C. and Oliveira, S.C.

Notes: To test the feasibility of the Mycobacterium bovis Ag85B gene as a DNA vaccine, the gene was amplified by PCR, introducing EcoRI and XbaI restriction endonucleases onto the primers, digested with the restriction enzymes and ligated into the pCI Mammalian Expression Vector. After transformation, the constructs were isolated using the Wizard® Plus Minipreps DNA Purification System and analyzed using endonuclease digestion and DNA sequencing. To confirm production of a maltose binding protein (MBP)-Ag85B fusion protein in E. coli, the cell lysate was analyzed by Western blot, stained with a rabbit anti-MBP serum and secondary antibody Anti-Rabbit IgG (Fc), AP Conjugate (1:10,000 dilution) and developed with BCIP/NBT. BALB/c mice pretreated with 10mM cardiotoxin prior to intramuscular DNA immunization using construct pCI-Ag85B, administered at days 0, 15, 30, and 45. The immunization course was 50µl of DNA at a concentration of 1µg/µl in PBS with each animal receiving a total of 100µg of plasmid DNA. Antibody response was assessed at 15 days with an ELISA assay and cytokine presence tested two weeks after immunization using splenocytes for ELISA assays and ICC. (3552)

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Nucl. Acids Res. 34, e7. Four-base codon mediated mRNA display to construct peptide libraries that contain multiple nonnatural amino acids. 2006

Muranaka, N., Hohsaka, T. and Sisido, M.

Notes: The authors devised an mRNA display system to generate a peptide library with multiple nonnatural amino acids incorporated into the proteins, an important feature of peptide libraries for successful drug discovery. An mRNA with 3 four-base codons at a random position was used as a template in an in vitro translation system in the presence of charged tRNAs carrying four-base codons. In vitro translations were performed using 3.6 × 1013 molecules of mRNA template and the E. coli S30 Extract System. The mRNA template contained a T7 tag sequence, so the translation products could be detected using an anti-T7 tag antibody and the Anti-Mouse IgG (H+L), AP Conjugate. The mRNA-displayed peptides also incorporated a polyhistidine tag so that they could be purified using the MagneHis™ Ni-Particles. After selecting for the desired protein characteristic, the mRNA portion of the mRNA-displayed peptides was reverse transcribed and quantitated by real-time PCR. PCR products were cloned into the pGEM®-T Vector prior to sequencing. (3651)

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Toxicol. Sci. 85, 727-734. Inhibitory effects of vitamin A on TCDD-induced cytochrome P-450 1A1 enzyme activity and expression. 2005

Yang, Y.M., Huang, D.Y., Liu, G.F., Zhong, J.C., Du, K., Li, Y.F. and Song. X.H.

Notes: The ability of the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to upregulate cytochrome P450 CYP1A1 levels in mouse liver was examined. Total mouse liver RNA was reverse transcribed using the Reverse Transcription System, and the resulting CYP1A1 cDNA was quantitated using real-time PCR. CYP1A1 protein levels were quantitated by Western blot using an anti-CYP1A1 antibody, the Anti-Rabbit IgG (Fc), Alkaline Phosphatase Conjugate secondary antibody and the Western Blue® Stabilized Substrate for Alkaline Phosphatase.
(3443)

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Nat. Neurosci. 3, 323-29. Synapsins as mediators of BDNF-enhanced neurotransmitter release. 2000

Jovanovic, J.N., Czernik, A.J., Fienberg, A.A., Greengard, P., and Sihra, T.S.

Notes: Treatment of  brain-derived neurotrophic factor increased MAPK-dependent synapsin I phosphorylation in rat cerebral cortex synaptosomal preparations. MAPK activity was determined by Western blot analysis using the Anti-ACTIVE® MAPK (1:10,000 dilution) pAb to detect the dually phosphorylated forms of MAPK. CaM kinase II activity was assayed by Western blot analysis with the Anti-ACTIVE® CaM KII pAb, Rabbit, (pT286) (1:2500 dilution). The Anti-Mouse IgG (H+L), AP Conjugate (1:1000 dilution) was used as a secondary antibody in Western blot analysis to detect TrkB expression. (2393)

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Proc. Natl. Acad. Sci. USA 96, 2970-2975. Mutator phenotypes of yeast strains heterozygous for mutations in the MSH2 gene. 1999

Drotschmann, K., Clark, A.B., Tran, H.T., Resnick, M.A., Gordenin, D.A., Kunkel, T.A.

Notes: Immunoblots, on PVDF, were developed with the Anti-Rabbit IgG Alkaline Phosphatase Conjugate and the Western Blue® Stabilized Substrate for Alkaline Phosphatase. (1200)

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Genetics 150, 1639-1648. Molecular consequences of Ds insertion into and excision from the helix- loop-helix domain of the maize R gene. 1998

Liu, Y., Wang, L., Kermicle, J.L., Wessler, S.R.

Notes: Poly(A)+ RNA was purified with PolyATtract® mRNA Isolation Systems. Anti-Rabbit IgG AP Conjugate, and BCIP/NBT were used in Western blotting. R transcripts were synthesized from the Lc cDNA in the pGEM-7Z(+) Vector, and were used in in vitro translation by Rabbit Reticulocyte Lysate Systems to be used as a positive control in Western. (0781)

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Biotechnol. Appl. Biochem. 28, 125-131. Recombinant human mast cell tryptase beta: stable expression in Pichia pastoris and purification of fully active enzyme. 1998

Niles, A.L., Maffitt, M., Haak-Frendscho, M., Wheeless, C.J., and Johnson, D.A.

Notes: Human mast cell tryptase was expressed and purified from Pichia pastoris by hydrophobic interaction chromatography followed by affinity chromatography on immobilized heparin. A faint band at 33Da and diffuse bands at 34.2, 35.9 and 50 kDa were observed on SDS/PAGE due to differences in the post-translational glycosylation of asparagine residues. Expression patterns of human tryptase were analysed by separating proteins by 10% SDS/PAGE , transferring to nitrocellulose membrane, and immunoblotting with the Anti-Human Tryptase mAb Biotin at 100 ng/ml in TBST. The blots were incubated with a 1:5000 dilution of Promega's Anti-Mouse IgG (H+L), AP Conjugate and developed with the Western Blue® Stabilized Substrate for Alkaline Phosphatase. (2451)

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