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Author (up) Levine, R.L.; Garland, D.; Oliver, C.N.; Amici, A.; Climent, I.; Lenz, A.G.; Ahn, B.W.; Shaltiel, S.; Stadtman, E.R. file  url
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  Title Determination of carbonyl content in oxidatively modified proteins Type Journal Article
  Year 1990 Publication Methods in Enzymology Abbreviated Journal Methods Enzymol  
  Volume 186 Issue Pages 464-478  
  Keywords  
  Abstract This chapter discusses methods to determine carbonyl content in oxidatively modified proteins. The methods described are (1) reduction of the carbonyl group to an alcohol with tritiated borohydride; (2) reaction of the carbonyl group with 2,4-dinitrophenylhydrazine to form the 2,4-dinitrophenylhydrazone; (3) reaction of the carbonyl with fluorescein thiosemicarbazide to form the thiosemicarbazone; and (4) reaction of the carbonyl group with fluorescein amine to form a Schiff base followed by reduction to the secondary amine with cyanoborohydride. Van Poelje and Snell have also quantitated protein-bound pyruvoyl groups through formation of a Schiff base with p-aminobenzoic acid followed by reduction with cyanoborohydride. Although a systematic investigation has not appeared, this method should also be useful in detecting other protein-bound carbonyl groups. Carbonyl content of proteins is expressed as moles carbonyl/mole subunit for purified proteins of known molecular weight. For extracts, the results may be given as nanomoles carbonyl/milligram protein. For a protein having a molecular weight of 50,000, a carbonyl content of 1 mol carbonyl/mol protein corresponds to 20 nmol carbonyl/mg proteins.

Subject headings: Amino Acids/analysis; Animals; Borohydrides; Glutamate-Ammonia Ligase/metabolism; Glutamates/*analysis; Hydrolysis; Indicators and Reagents; Oxidation-Reduction; *Proteins/isolation & purification/metabolism; Tritium
 
  Call Number Serial 2269  
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Author (up) McPheeters, D.S.; Wise, J.A. url  openurl
  Title Measurement of in vivo RNA synthesis rates Type Journal Article
  Year 2013 Publication Methods in Enzymology Abbreviated Journal Methods Enzymol  
  Volume 530 Issue Pages 117-135  
  Keywords Gene Expression Regulation, Fungal; RNA, Fungal/*genetics; Saccharomyces cerevisiae/*genetics; Schizosaccharomyces/*genetics; Transcription, Genetic; Immobilized DNA/RNA; Immobilized probes; In vivo RNA synthesis rates; Labeled RNA; Nascent transcripts  
  Abstract A technique is described to directly measure ongoing transcription from individual genes in permeabilized cells of either the budding yeast Saccharomyces cerevisiae or the fission yeast Schizosaccharomyces pombe. Transcription run-on (TRO) analysis is used to compare the relative rates of synthesis for specific transcripts in cells grown under different environmental conditions or harvested at different stages of development. As the amount of an individual RNA species present at any given time is determined by its net rate of synthesis and degradation, an accurate picture of transcription per se can be obtained only by directly measuring de novo synthesis of RNA (if you are interested in RNA degradation, see Method for measuring mRNA decay rate in Saccharomyces cerevisiae). Most techniques employed to measure changes in the relative levels of individual transcripts present under different conditions, including Northern analysis (see Northern blotting), RT-PCR (see Reverse-transcription PCR (RT-PCR)), nuclease protection assays (see Explanatory Chapter: Nuclease Protection Assays), and genome-wide assays, such as microarray analysis and high throughput RNA sequencing, measure changes in the steady-state level of a transcript, which may or may not reflect the actual changes in transcription of the gene. Recent studies carried out in fission yeast have demonstrated that increases in the steady-state level (accumulation) of many individual mRNAs occur without any significant changes in transcription rates (McPheeters et al., 2009), highlighting the important role of regulated RNA stability in determining gene expression programs (Harigaya et al., 2006).  
  Call Number Serial 1345  
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Author (up) Pace, C.N. file  url
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  Title Determination and analysis of urea and guanidine hydrochloride denaturation curves Type Journal Article
  Year 1986 Publication Methods in Enzymology Abbreviated Journal Methods Enzymol  
  Volume 131 Issue Pages 266-280  
  Keywords Guanidine; *Guanidines; Kinetics; *Protein Conformation; *Protein Denaturation; Spectrometry, Fluorescence; *Urea  
  Abstract  
  Call Number Serial 259  
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