Nuclease activity assay

The synthetic sugar substrate is colourless but the p-nitrophenol is formed as the green-yellow coloured p-nitrophenate anion that has an absorption maximum at about 400 nm. Similar assay procedures can be used to determine nuclease activity with DNase I ... 

The fluorogenic 5 nuclease assay, which utilises the 5 nuclease activity inherent as a secondary function of Taq DNA polymerase to cleave a gene-specific probe, thereby releasing a fluorescent molecule for detection. 

Flow cytometer-based assays for several other apoptotic indicators have been developed. Changes in intracellular ion levels, in particular Ca + and H+, are considered good early indicators of compound-induced cellular toxicity. Elevated levels may be important in apoptosis by activating nuclease activity. Intracellular pH and levels are... 

This chapter will detail protocols used by the authors to study biochemical aspects of the plant polyadenylation complex subunits [8-11]. Three sections are presented for this. The first deals with approaches for overexpression and purification of tagged proteins produced in E. coli. The second describes the suite of electrophoretic and associated methods used to assess protein quality and the outcomes of particular biochemical reactions or assays. The third section describes a set of assays that can be performed using the same lysates, purified proteins, and electrophoretic methods to assess protein-protein interactions, RNA binding capability, and nuclease activity. 

Nuclease activity is assayed by measuring the release of acid-soluble products from heat-denatured DNA (e.g., calf thymus). A typical assay mixture (0.5 ml) contains 30 mM NaAc (pH 4.6), 50 mM NaCl, 1 mM zinc acetate, 5% (v/v) glycerol, 0.5 mg/ml heat-denatured DNA, and enzyme. The assay mixture is incubated for 10 min at 37°C. 

Note The typical assay just described is not reliable in measuring polymerase activity in crude cell extracts. More critically, the assay cannot be applied to polymerases lacking 5 -(exo)nuclease activity. A polymerase may better be assayed uniquely by its ability to catalyze a 5 3 strand synthesis reaction. One such... 

The application of directed evolution approaches for the change or extension of the specificity of a restriction enzyme is hampered by the fact that an in vivo selection assay is not available and that examination of endonuclease activity in vitro usually requires purification of the enzymes to avoid background activity by other nucleases prevalent in all cells. This means that an altered or extended specificity can only be observed with sufficiently purified protein preparations, thereby unfortunately separating genotype and phenotype. As neither a reliable in vivo test nor the secretion of restriction endo-... 

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