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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Additional info for Methods of Biochemical Analysis, Volume 20
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 ) .
Methods of Biochemical Analysis, Volume 20