Primary-secondary phosphate buffer

FIGURE 5.26. Antigen-antibody construction of a monolayer glucose oxidase electrode with an attached ferrocenium cosuhstrate and cyclic voltammetric response in a phosphate buffer (pH 8) at 25°C and a scan rate of 0.04 V/s. a Attached ferrocene alone, h Addition of the substrate, c Primary plots, d Secondary plot. The numbers on the curves in parts h and c are the values of the substrate concentration in mM. Adapted from Figure 2 in reference 24, with permission from the American Chemical Society. 

The sections are incubated in a primary antibody (diluted appropriately) for 72 hr at 4°C in a sealed humid chamber the incubation is carried out by applying droplets of the antibody to the sections. After being rinsed in the buffer, the sections are incubated for 90min in secondary antiserum diluted 1 50 with PBX (0.3% Triton X-100, 0.01% sodium azide and 0.1 M phosphate buffer) and then treated for 1 hr under agitation in peroxidase-antiperoxidase (PAP), diluted 1 100 with PBX, in a sealed humid chamber in both cases. 

Haga et al. (1983) have measured the ORD and CD spectra of as2-casein in Ca2+-free phosphate buffer, pH 7.2, and estimated the content of a helix to be about four times as large as in aSi-casein, which they attribute to the lower proline content of the more phosphorylated protein. There are marked differences in the primary and predicted secondary structures of aS2-type caseins from different species. One notable constant feature, however, is a predicted a helix... 

Fig. 6 Cyclic voltammetric analysis of the kinetics of an electrode coated with antigen-antibody immobilized monomolecular layer of redox enzyme with a one-electron reversible cosubstrate in the solution, (a) Cyclic voltammetry at saturation coverage (2.6 x 10 mol cm ) of glucose oxidase with 0.1 M glucose and 0.1 mM ferrocenemethanol in a pH 8 phosphate buffer (0.1 M ionic strength). The dotted and dashed lines represent the cyclic voltammogram (0.04 V sec ) in the absence and presence of glucose (0.1 M), respectively. The full line represents the catalytic contribution to the current,/ cat (see text), (b) Primary plots obtained under the same conditions with, from top to bottom, 0.01, 0.02, 0.05, and 0.1 M glucose, (c) Secondary plot derived from the intercepts of the primary plots in (b).

Teichert et al. [45] were the first to carry out TLC of amines. The hydrochlorides were dissolved in 70% alcohol and applied in 1 to 10 [xg amounts. The hi /-values and experimental conditions for the separation are summarised in Table 94. As can be seen from the table, only a partial improvement in separation is achieved by buffering silica gel G layers with a mixture of 0.2M primary potassium phosphate and 0.2 M secondary sodium phosphate (1 -h 1) or with 0.15M sodium acetate solution. 

I = Silica gel G layer, buffered to pH 6.8 (25 g silica gel G slurried with 50 ml of a mixture of equal amounts of 0.2 M primary potassium phosphate and 0.2M secondary sodium phosphate solutions). Chloroform-96% ethanol (90 + 10) as solvent. 

Vincristine (VC) and vinblastine (VB) are dimeric catharanthus alkaloids isolated from the plant Catharanthus roseus. A capillary zone electrophoresis (CZE) was conducted for systematic and comprehensive study of the separation and quantification of two dimeric catharanthus alkaloids. Various separation parameters such as buffer concentration and pH, column internal diameter, and applied voltage were studied column, 72 cm (57 cm effective length) x 75 pm I.D. buffer, 0.2 M ammonium acetate solution, pH 6.2 and an applied voltage of 10 kV. Although the separation of VB and VC was the primary focus, the separation parameters determined in this study can be applied to the separation of other alkaloids as well. Separation of other alkaloids in the plant samples was observed under conditions presented in this work. A secondary objective of this study was to develop a method with experimental conditions which could be applied to electrophoresis-mass spectrometry. For this purpose, ammonium acetate buffers, which are more compatible with mass spectrometry than the widely used phosphate buffers, were used exclusively. Also, methanol-water-acetic acid was used as external buffer for the same reason [16]. 

Apart from treating OA of the knee, HA has been used for the treatment of chronic shoulder pain associated with glenohumeral osteoarthritis (GH-OA) [33 ]. This study evaluates the safety and efficacy of HA in treating GH-OA, by employing a double-blind, randomised, controlled, multicentre trial in which 300 patients with GH-OA were eruoU 150 patients received HA and 150 received phosphate-buffered saline (PBS) in three weekly injections and were evaluate over 26 weeks. Primary and secondary outcome measurements were VAS for pain and the percentage of OMERACT-OARSI high responders. 

We hydrolyzed ATP and ADP in 1 N and 0.1 N HC1 and in buffered solutions at pH 4j nd 8 in which the hydrolysis medium was variously enriched in °0 to either 10% or 20%. To assess the isotopic enrichment of each such solution for use in the nucleotide hydrolysis experiments, we hydrolyzed PCI, in the solution, esterified the resultant phosphoric acid/inorganic phosphate (P.) by reaction with diazomethane, and determined the isotopic distribution of the trimethyl phosphate (TMPO) by mass spectrometry. The 1 N and 0.1 N HC1 hydrolyses were allowed to proceed for 45 min and 10 hr, respectively, at 70, insuring complete conversion of ATP into AMP + 2P. The pH 8 hydrolyses were allowed to proceed for 36 hr at 70 to a point (20-25% completion) at which the ratio of ADP to AMP established that 96% and 4%, respectively, of the P. released had arisen by the primary and secondary hydrolysis steps, namely, ATP ADP + P. and ADP " AMP + P. 0The pH 4 hydrolyses were allowed to proceed for 24 hr, also at 70, to 40% completion. 

APases hydrolyze numerous phosphate esters, such as those of primary and secondary alcohols, phenols and amines (Levine, 1974). One unit of activity of APase corresponds to the hydrolysis of 1.0 pmole of p-nitrophenyl phosphate (p-NPP) per min (in 100 mM glycine, 1 mM ZnCb, 1 mM MgCl2 and 6 mM p-NPP, pH 10.4 or in 1 M diethanolamine, 0.5 mM MgCU and 15 mM p-NPP, pH 9.8). The bovine enzyme generally has a specific activity of 1000 and 2000 U/mg in these two buffers, respectively, at 37°C. At 25°C, activity is reduced to about half. This demonstrates that buffers may have a marked influence on the enzymatic activity of APases which explains the great differences in activity given for commercial preparations. Assays with p-NPP above 30°C suffer from the spontaneous hydrolysis of this substrate, with serious consequences for the enzyme kinetics (see below). The bacterial enzyme has lower activity than the bovine intestinal enzyme. 

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