ISBN-10: 0470110392

ISBN-13: 9780470110393

ISBN-10: 0471307556

ISBN-13: 9780471307556

An encyclopedia of equipment in biochemical research, this contemporary sequence retains biochemists and analytical chemists abreast of experimental techniques and enhancements in biochemical innovations and instrumentation.

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The volume of the cycled reaction analyzed fluorometrically is dependent upon the sensitivity of the instrumental setting and, of course, on the concentration of gluc-6-P present. Ordinarily, about 5 to 10 pl of the Step I11 reaction is analyzed a t a low-to-medium sensitivity setting on the fluorometer. ANALYSIS OF CYCLIC 3’,5’-ADENOSINE MONOPHOSPHATE 27 E. INTERPRETATION OF RESULTS The concentration of gluc-6-P in the aliquot of each sample carried through the entire procedure without phosphodiesterase treatment [ (-) PD blank] is subtracted from the average concentration present in the two aliquots exposed to this enzyme.

Procedures. 0, 3mM MgSO, and 1 mM EDTA. This volume of reagent very efficiently rinses the surface of the 5 ml conical centrifuge tube with a vortex-action mechanical mixer. 0 added to only two of the three samples. The tops of the tubes are then covered with a sheet of aluminum foil and incubated a t 3 7 O for 60 min. The reaction is terminated by placing the tubes in a 90° water bath for 4 min. c. Preliminary Procedures and Precautions. 96 mg/ml Myokinase,ac 40 pglml; pyruvate kinase,ac 250 pg/ml Bovine heart phosphodiesterase,d 11 r g h l Enzymes 1000 45 40 25 Reaction volume (~1) 3min 1 hr 17 hrs 60 min Time 25" 37" 26" 37" 2 min a t 90" 0" ice bath 4 min a t 90" Temper- Method of ature termination Incubation a Concentrations in the reagent are given.

In order to comprehend fully the principle of these methods, a clear understanding of the general properties of the EB-nucleic acid reaction is needed. These properties are summarized in the first part of this chapter. 11. SUMMARY OF THE PROPERTIES OF THE ETHIDIUM BROMIDE NUCLEIC ACID REACTION E B binds to DNA or RNA a t two different kinds of site. The first and more important, called the “fluorescent site,” is the one for which E B has the strongest affinity. 1. Properties of the First Binding Site (Fluorescent) When E B binds to this site, and only to this site, a large and specific increase of its fluorescence is observed ( 2 6 2 7 ) .

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Methods of Biochemical Analysis, Volume 20


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