Neutral buffers

In the case of 2- and 6-hydroxypteridine and their derivatives, the anhydrous species in neutral solutions (produced by rapid addition of equilibrated alkaline solutions to neutral buffers) change sufficiently slowly into the hydrated species that serial scans on a recording spectrophotometer can be used to demonstrate the process. The results shown in Fig. 1 for 6-hydroxy-2-methylpteridine are typical. 

In systems such as the 2- and 6-hydroxypteridines, sudden addition of an alkaline solution to a neutral buffer, or of a neutral solution to an alkaline buffer, displaces the equilibrium between hydrated and anhydrous species (because the anions are less hydrated than the neutral molecules). By measuring the time-dependent change of optical density at a selected wavelength, a first-order rate constant, obs5 can be obtained. This constant is a composite one, and to see its relationship to other quantities some discussion is necessary. 

It is the rapid increase in rates of hydration with increasing hydrogen ion concentration that prevents measurement with existing apparatus of the -pKa values of anhydrous bases such as pteridine. For example, at pH 1, hydration of the anhydrous cation is half-complete in 0.01 sec at 20°. Conversely, it is the comparative slowness of the reactions in near-neutral solutions that makes it possible, by adding acid solutions to near-neutral buffers, using the stopped-flow technique, to determine the p STa values of the hydrated species. 

According to the Kuwabara-Wassink paper, the purified luciferin in aqueous neutral buffer solution showed an absorption maximum at 320 nm, and a fluorescence emission peak at 490 nm. The luminescence emission maximum measured with Airth s fungal luciferase system was 524 nm at pH 6.5, whereas the chemiluminescence emission maximum of the luciferin with H2O2 plus a droplet of strong NaOH plus ferrous sulfate was 542 nm. No information was reported on the chemical nature of the luciferin. 

Coelenterazine and the corresponding luciferase can be easily tested in the field. A small piece of tissue sample is put in a test tube with methanol (for coelenterazine) or water (for luciferase), and crushed with a spatula. To measure coelenterazine, a buffer solution containing a coelenterazine luciferase is injected into a small amount of the fluid part of the crushed sample mixture. Similarly, luciferase can be measured with a buffer solution containing coelenterazine. The presence of Cypridina luciferin can be tested in the same fashion, with the methanol extract of samples and crude Cypridina luciferase. However, the detection of a very weak Cypridina luciferase activity in the field is not recommended (see Section C5.6). To test the presence of a Ca2+-sensitive photoprotein, crush a sample in a neutral buffer solution containing 20-50 mM EDTA, and then add lOmM calcium acetate to a small portion of the fluid part of the crushed sample to detect any light emission. 

Prepare a 1 40 dilution of stock Giemsa stain in neutral buffered water, pH 7.0 to 7.2 (generally, 2 ml of Giemsa stock plus 38 ml of buffered water with 0.01% Triton X-100). 

Prepare a 1 50 dilution of stock Giemsa stain in neutral buffered water (pH 7.0 to 7.2). 

Compound (Miles Laboratories, Elkhart, IN), snap-frozen, and cut into sections for comparison with paraffin-embedded cell sections (3) FFPE Cell Blocks Six cell pellets were fixed in 10% neutral buffered formalin immediately after harvest, at room temperature for 6,12,24h, 3,7, and 30 days, respectively. For further comparison with the cell model system, recently collected sample of human breast cancer tissues were processed by OCT-embedding and snap-freezing the corresponding routine FFPE block that was obtained from the Norris Cancer Hospital and Research Institute at the University of Southern California Keck School of Medicine (USC). This tissue block was processed routinely (formalin-fixed 24h and processed by automatic equipment). 

Figure 12.1 Typical good quality specimen. Small intestine fixed for 24 h in neutral buffered formalin after grossing at 2 mm. Most nuclei show good chromatin patterns, but cell membranes are indistinct. 

The successful of recovery of RNase A functional activity by a heat-induced AR method suggested the possibility of recovering RNase A immunoreactivity as well. The immunoreactivity of native RNase A and RNase A that was incubated at a concentration of 4 mg/mL in 10% neutral buffered formalin for 1 day and then freed of formaldehyde by dialysis against PBS was compared using capture enzyme-linked immunosorbent assay (ELISA). Selected fractions that... 

Figure 15.10 SDS-PAGE of native RNase A (lane 1) and RNase A incubated in 10% neutral buffered formalin for 9 days (lane 2) or in 5% neutral buffered formalin for 1 day (lane 4). The formalin-treated samples were then demodified for4h inTAE buffer (pH 4) at 65°C 10% formalin oligomers (lane 3) and 5% formalin oligomers (lane 5). M, molecular mass markers in kDa. See Rait et al.11 for details.

Tapioca and maize amylopectins have been sub-fractionated by fractional precipitation from aqueous solution with increasing amounts of methanol,64 71 and potato amylopectin by preferential precipitation on electrodialysis of the iodine complex.72 When these three amylopectins were subjected to chromatography, and eluted with a neutral buffer, all were found to consist of several sub-fractions.70... 

In Chapter 8, you learned that buffers resist changes in pH when acids or bases are added. Buffers have many uses in a chemistry lab. For example, commercial buffers with certified pH values are available. Chemists in analytical laboratories use these acidic, basic, and neutral buffers to ensure the accuracy of their pH meters across the pH range. 

Fixation Fix tissue in 10% neutral buffered formalin (or equivalent) by placing slides in a shde rack and incubating them in a staining dish containing the fixative. Upon removal from formahn solution, rinse with tap and double distilled water (ddH20) to remove salts. 

Perhaps the most important parameter involved in aqueous-organic mixtures is their effective protonic activity (denoted by pH or pan). This parameter has been measured for most commonly used buffers in all selected mixtures down to their freezing point (Hui Bon Hoa and Douzou, 1975 Douzou ei al., 1976). Values of pH depend on solvent and temperature in a way that varies for different buffers, but with the data available a medium of known pH under any desired condition may be prepared. An example of the effect of solvent and temperature is provided by Tris-HCl buffer a solution of this at pH 8.0 in water at 20 C will be pH 10.5 in 50% (v/v) ethanediol at -40 C (Douzou et al, 1976). On the other hand, neutral buffers such as phosphate undergo... 

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