The Experts below are selected from a list of 33 Experts worldwide ranked by ideXlab platform

Rodrigo C. Rossi - One of the best experts on this subject based on the ideXlab platform.

Fernando Coelho - One of the best experts on this subject based on the ideXlab platform.

Elizabeth M. Wilson - One of the best experts on this subject based on the ideXlab platform.

  • 8 use of fluorescent Chloramphenicol Derivative as a substrate for Chloramphenicol acetyltransferase assays
    Recombinant DNA Methodology II, 1995
    Co-Authors: Dennis E. Hruby, Elizabeth M. Wilson
    Abstract:

    Publisher Summary This chapter discusses the use of fluorescent Chloramphenicol Derivatives. The DNA sequences encoding bacterial Chloramphenicol acetyltransferase (CAT) enzyme is fused to heterologous transcriptional regulatory signals and used as a “reporter gene” for measuring the rate of transcription of chimeric genes, as well as the translation and/or stability of the chimeric transcripts within the context of transformed bacterial cells, transfected tissue culture cells, or genetically engineered recombinant viruses. The chapter presents the two types of CAT assay procedures that are currently available. The first involves using Chloramphenicol as the substrate for acetylation. The acetylated Derivatives are resolved from the substrate by thin-layer chromatography and quantitated by autoradiography and subsequent densitometric scanning or by eluting the acetylated Derivatives from the plate followed by scintillation counting. The second method utilizes acetyl-Coenzyme A as the substrate. In this case, the acetylated reaction products are collected by organic extraction and quantitated directly by scintillation counting. The chapter additionally discusses the advantages of using a fluorescent Chloramphenicol substrate to assay CAT activity. The assay is rapid and easily quantified and the results can be evaluated immediately. No radioactive materials are required. This dispenses with the need for film, fluors, or scintillation counters and disposal of radioactive wastes.

  • [8] – Use of Fluorescent Chloramphenicol Derivative as a Substrate for Chloramphenicol Acetyltransferase Assays
    Recombinant DNA Methodology II, 1995
    Co-Authors: Dennis E. Hruby, Elizabeth M. Wilson
    Abstract:

    Publisher Summary This chapter discusses the use of fluorescent Chloramphenicol Derivatives. The DNA sequences encoding bacterial Chloramphenicol acetyltransferase (CAT) enzyme is fused to heterologous transcriptional regulatory signals and used as a “reporter gene” for measuring the rate of transcription of chimeric genes, as well as the translation and/or stability of the chimeric transcripts within the context of transformed bacterial cells, transfected tissue culture cells, or genetically engineered recombinant viruses. The chapter presents the two types of CAT assay procedures that are currently available. The first involves using Chloramphenicol as the substrate for acetylation. The acetylated Derivatives are resolved from the substrate by thin-layer chromatography and quantitated by autoradiography and subsequent densitometric scanning or by eluting the acetylated Derivatives from the plate followed by scintillation counting. The second method utilizes acetyl-Coenzyme A as the substrate. In this case, the acetylated reaction products are collected by organic extraction and quantitated directly by scintillation counting. The chapter additionally discusses the advantages of using a fluorescent Chloramphenicol substrate to assay CAT activity. The assay is rapid and easily quantified and the results can be evaluated immediately. No radioactive materials are required. This dispenses with the need for film, fluors, or scintillation counters and disposal of radioactive wastes.

  • Use of fluorescent Chloramphenicol Derivative as a substrate for Chloramphenicol acetyltransferase assays.
    Methods in Enzymology, 1992
    Co-Authors: Dennis E. Hruby, Elizabeth M. Wilson
    Abstract:

    Publisher Summary This chapter discusses the use of fluorescent Chloramphenicol Derivative as a substrate for Chloramphenicol acetyltransferase assays. The DNA sequences encoding bacterial Chloramphenicol acetyltransferase (CAT) enzyme are commonly fused to heterologous transcriptional regulatory signals, and used as a “reporter gene” for measuring the rate of transcription of chimeric genes, as well as the translation and/or stability of the chimeric transcripts within the context of transformed bacterial cells, transfected tissue culture cells, or genetically engineered recombinant viruses. CAT activity has a number of advantages over the traditional methods: (1) No radioactive materials are required. This dispenses with the need for film, fluors, scintillation counters, and disposal of radioactive wastes; (2) the assay is rapid and easily quantified; (3) the results can be evaluated immediately. Taken together, these results would suggest that the use of a fluorescent Chloramphenicol substrate provides an attractive alternative method for measuring CAT activity in the extracts of cells, which express this enzyme. The development of a modified CAT enzyme assay that uses fluorescent Chloramphenicol substrates promises to enhance the utility of this system by eliminating the use of radioactive materials.

Dennis E. Hruby - One of the best experts on this subject based on the ideXlab platform.

  • 8 use of fluorescent Chloramphenicol Derivative as a substrate for Chloramphenicol acetyltransferase assays
    Recombinant DNA Methodology II, 1995
    Co-Authors: Dennis E. Hruby, Elizabeth M. Wilson
    Abstract:

    Publisher Summary This chapter discusses the use of fluorescent Chloramphenicol Derivatives. The DNA sequences encoding bacterial Chloramphenicol acetyltransferase (CAT) enzyme is fused to heterologous transcriptional regulatory signals and used as a “reporter gene” for measuring the rate of transcription of chimeric genes, as well as the translation and/or stability of the chimeric transcripts within the context of transformed bacterial cells, transfected tissue culture cells, or genetically engineered recombinant viruses. The chapter presents the two types of CAT assay procedures that are currently available. The first involves using Chloramphenicol as the substrate for acetylation. The acetylated Derivatives are resolved from the substrate by thin-layer chromatography and quantitated by autoradiography and subsequent densitometric scanning or by eluting the acetylated Derivatives from the plate followed by scintillation counting. The second method utilizes acetyl-Coenzyme A as the substrate. In this case, the acetylated reaction products are collected by organic extraction and quantitated directly by scintillation counting. The chapter additionally discusses the advantages of using a fluorescent Chloramphenicol substrate to assay CAT activity. The assay is rapid and easily quantified and the results can be evaluated immediately. No radioactive materials are required. This dispenses with the need for film, fluors, or scintillation counters and disposal of radioactive wastes.

  • [8] – Use of Fluorescent Chloramphenicol Derivative as a Substrate for Chloramphenicol Acetyltransferase Assays
    Recombinant DNA Methodology II, 1995
    Co-Authors: Dennis E. Hruby, Elizabeth M. Wilson
    Abstract:

    Publisher Summary This chapter discusses the use of fluorescent Chloramphenicol Derivatives. The DNA sequences encoding bacterial Chloramphenicol acetyltransferase (CAT) enzyme is fused to heterologous transcriptional regulatory signals and used as a “reporter gene” for measuring the rate of transcription of chimeric genes, as well as the translation and/or stability of the chimeric transcripts within the context of transformed bacterial cells, transfected tissue culture cells, or genetically engineered recombinant viruses. The chapter presents the two types of CAT assay procedures that are currently available. The first involves using Chloramphenicol as the substrate for acetylation. The acetylated Derivatives are resolved from the substrate by thin-layer chromatography and quantitated by autoradiography and subsequent densitometric scanning or by eluting the acetylated Derivatives from the plate followed by scintillation counting. The second method utilizes acetyl-Coenzyme A as the substrate. In this case, the acetylated reaction products are collected by organic extraction and quantitated directly by scintillation counting. The chapter additionally discusses the advantages of using a fluorescent Chloramphenicol substrate to assay CAT activity. The assay is rapid and easily quantified and the results can be evaluated immediately. No radioactive materials are required. This dispenses with the need for film, fluors, or scintillation counters and disposal of radioactive wastes.

  • Use of fluorescent Chloramphenicol Derivative as a substrate for Chloramphenicol acetyltransferase assays.
    Methods in Enzymology, 1992
    Co-Authors: Dennis E. Hruby, Elizabeth M. Wilson
    Abstract:

    Publisher Summary This chapter discusses the use of fluorescent Chloramphenicol Derivative as a substrate for Chloramphenicol acetyltransferase assays. The DNA sequences encoding bacterial Chloramphenicol acetyltransferase (CAT) enzyme are commonly fused to heterologous transcriptional regulatory signals, and used as a “reporter gene” for measuring the rate of transcription of chimeric genes, as well as the translation and/or stability of the chimeric transcripts within the context of transformed bacterial cells, transfected tissue culture cells, or genetically engineered recombinant viruses. CAT activity has a number of advantages over the traditional methods: (1) No radioactive materials are required. This dispenses with the need for film, fluors, scintillation counters, and disposal of radioactive wastes; (2) the assay is rapid and easily quantified; (3) the results can be evaluated immediately. Taken together, these results would suggest that the use of a fluorescent Chloramphenicol substrate provides an attractive alternative method for measuring CAT activity in the extracts of cells, which express this enzyme. The development of a modified CAT enzyme assay that uses fluorescent Chloramphenicol substrates promises to enhance the utility of this system by eliminating the use of radioactive materials.

Rc Rossi - One of the best experts on this subject based on the ideXlab platform.