Non-radioactive labeling of RNA transcripts<i>in vitro</i>with the hapten digoxigenin (DIG); hybridization and ELISA-based detection

Höltke, Hans-Joachim; Keßler, Christoph

doi:10.1093/nar/18.19.5843

Cited by 196 publications

(66 citation statements)

References 30 publications

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“…RNA probes were synthesized using ATP, GTP, CTP, UTP and digoxigenin (DIG)-labelled UTP together with SP6 or T7 RNA polymerase (Boehringer Mannheim, Germany). The vectors containing the human VH-and CH-region inserts were linearized with appropriate restriction enzymes and used as templates for in vitro transcription according to the method of Höltke & Kessler [43]. After transcription, the DNA templates were digested by treatment with RNAse-free DNAse I (Boehringer) for 15 min at 37ЊC, and the RNA probes were dissolved in hydrolysis buffer, containing 40 mm NaHCO 3 , 60 mm Na 2 CO 3 , pH 10.2.…”

Section: Cellsmentioning

confidence: 99%

Selective Usage of VH Genes in Adult Human B Lymphocyte Repertoires

et al. 1997

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Davidkova G, Pettersson S, Holmberg D, Lundkvist I. Selective Usage of VH Genes in Adult Human B Lymphocyte Repertoires. Scand J Immunol 1997;45:62-73 The authors have compared the VH gene utilization patterns among small resting immunocompetent B cells and large naturally activated B lymphocytes of healthy human adults. They employed a non-radioactive RNA in situ hybridization technique that allows detection of VH gene family expression at the single cell level. Pokeweed mitogen stimulated and unmanipulated mononuclear cells from peripheral blood and spleen of unrelated individuals were hybridized to digoxigenin-labelled antisense RNA probes specific for human VH families 1-6 and for the constant region genes Cm and Cg. The observed VH gene family utilization patterns did not correlate with the genomic complexity of human VH genes. The VH3 gene family was most frequently used among resting B cells in both peripheral blood and spleen. Among naturally activated lymphocytes the VH6 gene was markedly over-represented, while expression of the VH1 and VH3 gene families was decreased. The data show that V-region mediated selection participates in shaping the peripheral antibody repertoire in healthy adults.

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Section: Cellsmentioning

confidence: 99%

Selective Usage of VH Genes in Adult Human B Lymphocyte Repertoires

et al. 1997

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“…Seven-g aliquots were separated by denaturing gel electrophoresis (1.25% formaldehyde agarose gels; running buffer was 20 mM MOPS, 8 mM NaAc, and 1 mM EDTA) and electroblotted onto a nylon membrane. The following nonradioactive DIG filter hybridization and detection of RhoA mRNA was performed as described previously (36,37).…”

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confidence: 99%

Neosynthesis and Activation of Rho by Escherichia coli Cytotoxic Necrotizing Factor (CNF1) Reverse Cytopathic Effects of ADP-ribosylated Rho

Barth

Olenik

Sehr

et al. 1999

Journal of Biological Chemistry

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Clostridium botulinum exoenzyme C3 inactivates the small GTPase Rho by ADP-ribosylation. We used a C3 fusion toxin (C2IN-C3) with high cell accessibility to study the kinetics of Rho inactivation by ADP-ribosylation. In primary cultures of rat astroglial cells and Chinese hamster ovary cells, C2IN-C3 induced the complete ADP-ribosylation of RhoA and concomitantly the disassembly of stress fibers within 3 h. Removal of C2IN-C3 from the medium caused the recovery of stress fibers and normal cell morphology within 4 h. The regeneration was preceded by the appearance of non-ADP-ribosylated RhoA. Recovery of cell morphology was blocked by the proteasome inhibitor lactacystin and by the translation inhibitors cycloheximide and puromycin, indicating that intracellular degradation of the C3 fusion toxin and the neosynthesis of Rho were required for reversal of cell morphology. Escherichia coli cytotoxic necrotizing factor CNF1, which activates Rho by deamidation of Gln 63 , caused reconstitution of stress fibers and cell morphology in C2IN-C3-treated cells within 30 -60 min. The effect of CNF1 was independent of RhoA neosynthesis and occurred in the presence of completely ADP-ribosylated RhoA. The data show three novel findings; 1) the cytopathic effects of ADP-ribosylation of Rho are rapidly reversed by neosynthesis of Rho, 2) CNF1-induced deamidation activates ADP-ribosylated Rho, and 3) inhibition of Rho activation but not inhibition of Rho-effector interaction is a major mechanism underlying inhibition of cellular functions of Rho by ADP-ribosylation.The low molecular mass GTPases of the Rho family (Rho, Rac, and Cdc42) play key roles in the control of the actin cytoskeleton (1). In several cell lines, activation of RhoA induces formation of stress fibers and focal adhesions (2), whereas Rac and Cdc42 cause the formation of lamellipodia and filopodia, respectively (2-4). Moreover, Rho GTPases act as molecular switches in various signaling processes (5) including secretion (6), phagocytosis (7), endocytosis (8, 9), cell cycle progression (10), transcriptional activation (11) (22). C3 exoenzyme in particular is widely used as a cell biological tool to study the functions of Rho (23).Inasmuch as C3 exoenzyme appears to enter cells via nonspecific pinocytosis, its cell accessibility is poor (24). Recently, we constructed a C3 fusion toxin, which enters cells by using the binary C. botulinum C2 toxin as a carrier (25). The actin-ADP-ribosylating C2 toxin consists of the ϳ80-kDa binding component (C2II) and the 49-kDa enzymatic component (C2I) (26 -28). Both C2 components are separate proteins which assemble at the cell surface after C2II has bound to an unknown receptor. The C3 fusion toxin (C2IN-C3) contains the N-terminal domain (amino acid residues 1-225) of C2I (C2IN), which has no enzyme activity but binds to the C2II component and is sufficient for translocation of C2I into the cytosol. In contrast to C3 exoenzyme, the C2IN-C3 fusion toxin enters cells readily and causes the depolymerization of stress fibers at lo...

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“…(1)(2)(3) ELISA uses the principle of antigen-antibody reactions. By the ELISA method, it is possible to determine the concentration of an antigen, such as an enzyme, hormone or activation factor, in a sample solution.…”

Section: Introductionmentioning

confidence: 99%

Development of Wireless Electrical Conductivity Sensor Screening System to Evaluate Protein Binding to Sensor Films

2017

Sensors and Materials

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We developed a wireless electrical conductivity (EC) sensor screening system to evaluate changes in the EC of sensor films. This sensor system was designed to be equipped with a special sensor chip (14 × 40 mm 2 ), and changes in the EC of a droplet containing a sensor film (ϕ3 mm) on the chip were determined. Electrode pairs were fabricated on glass substrates by photolithography, and a well structure was formed around each electrode pair using photoreactive resin. This sensor system achieved parallel detection of EC signals from five wells, each containing 5 μl of solution with a sensor film in 10 s (N = 5). Furthermore, we developed sensor films that displayed the changes in the EC upon the adsorption and/or chemical binding of protein with Na + as a counterion. To form the sensor films, the surface of cellulose was chemically modified with alkylamino groups. The EC detected the sensor screening system using aminated cellulose with physically adsorbed bovine serum albumin (BSA) and the amount of Na + ions increased linearly with BSA concentration with a correlation coefficient (R 2 ) of 0.87. Meanwhile, aminated cellulose with chemically bound BSA through polylysine and glutaraldehyde and adsorbed Na + exhibited an EC that depended on the BSA concentration, with an R 2 value of 0.943. The detection limit of BSA was 10 ng/ml for both types of sensor film. On the basis of our results, the mechanism behind the increased sensitivity of the sensor films with immobilized protein in the presence of a counterion was proposed.

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Non-radioactive labeling of RNA transcriptsin vitrowith the hapten digoxigenin (DIG); hybridization and ELISA-based detection

Cited by 196 publications

References 30 publications

Selective Usage of VH Genes in Adult Human B Lymphocyte Repertoires

Selective Usage of VH Genes in Adult Human B Lymphocyte Repertoires

Neosynthesis and Activation of Rho by Escherichia coli Cytotoxic Necrotizing Factor (CNF1) Reverse Cytopathic Effects of ADP-ribosylated Rho

Development of Wireless Electrical Conductivity Sensor Screening System to Evaluate Protein Binding to Sensor Films

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