Showing papers on "Buffer solution published in 1995"

Antigen retrieval immunohistochemistry under the influence of pH using monoclonal antibodies.

Abstract: Antigen retrieval (AR) incorporating high-temperature microwave (MW) heating of tissue sections before immunostaining is a revolutionary technique that can unmask the antigens in formalin-fixed tissue sections, thus making them available for immunohistochemical staining. Although high temperature is believed to be the primary mechanism in retrieval of antigens, a variety of chemical solutions have been tested to define an optimal AR solution. We tested the hypothesis that pH of the AR solution may influence the quality of immunostaining by using seven different AR buffer solutions at a series of different pH values ranging from 1 to 10. We evaluated the staining of monoclonal antibodies to cytoplasmic antigens (AE1, HMB45, NSE), nuclear antigens (MIB-1, PCNA, ER), and cell surface antigens (MT1, L26, EMA) on routinely formalin-fixed, paraffin-embedded sections under different pH conditions with MW heating for 10 min. The intensity of immunostaining was graded in a blinded fashion. The pH value of the AR buffer solution was carefully measured before, immediately after, and 15 min after the AR procedure. The influence of pH on AR immunohistochemical staining can be summarized into three patterns. Some antigens (L26, PCNA, AE1, EMA, and NSE) showed excellent retrieval throughout the pH range. Other antigens (MIB1 and ER) showed strong intensity of immunohistochemical staining at very low pH and at neutral to high pH, but a dramatic decrease in the intensity of the AR immunostaining at moderately acidic pH (pH 3-6). Still others (MT1 and HMB45) showed increasing intensity of the AR immunostaining with increasing pH, but only weak immunostaining at low pH. Among the seven buffer solutions at any given pH value, the intensity of AR immunostaining was very similar. However, Tris-HCl buffer tended to produce better results at higher pH, compared with other buffers. Although high-temperature heating is believed to be the most important factor for the AR technique, the pH value of the AR solution is an important co-factor for some antigens. Optimization of the AR system should therefore include optimization of the pH of the AR solution. Our results indicate that AR immunostaining of Tris-HCl or sodium acetate buffer at pH 8-9 may be suitable for most antigens, although certain nuclear antigens show optimal staining at low pH.

Pressure-induced aggregation of β-lactoglobulin in ph 7.0 buffers

Abstract: Solutions of a β-lactoglobulin isolate in water, in potassium phosphate buffer (20 or 50 mmol/L), and in pressure-resistant buffers, at a protein concentration of 25 g/kg and at pH 7.0, were processed at 150, 250, 350 or 450 MPa and 25 °C, for 15 min, then stored at 4 °C, usually for 24 h, before analysis. Bis-Tris (20 or 50 mmol/L) and bis-Tris-propane (10, 20 or 50 mmol/L) were selected as pressure-resistant buffers, while the pH of water or phosphate buffer is known to decrease reversibly by 0.2–0.3 pH unit per 100 MPa. After pressurization, nitrogen solubility at pH 4.7 and 7.0, and aggregation patterns by polyacrylamide gel electrophoresis were determined. Both indicated that aggregation of β-lactoglobulin was more extensive in pressure-resistant buffers than in phosphate buffer or in water. Electrophoretic patterns also revealed the progressive formation of dimers to hexamers and of higher polymers of β-lactoglobulin as a function of the type and molarity of buffer and of the pressure level. All high molecular weight aggregates and most oligomers disappeared when pressurized solutions were treated with β-mercaptoethanol (15 mL/L) prior to electrophoresis. Thus pressure induced the formation of intermolecular S-S bonds, especially when the pH was maintained close to neutral. Previous studies with thermally-induced β-lactoglobulin gels had shown that SH/ S-S interchange reactions were enhanced at or above neutrality.

Electrocatalysis of Anodic Oxygen‐Transfer Reactions: The Electrochemical Incineration of Benzoquinone

Abstract: The electrochemical performance of several anode materials is compared for the electrochemical incineration of p-benzoquinone in acetate buffer media (pH 5). The chemical oxygen demand (COD) estimated for benzoquinone by titration with standard permanganate solution can be decreased to virtually zero by electrolysis at electrodes comprised of Fe(III)-doped {beta}-PbO{sub 2} films on Ti substrates. Carbon dioxide is a product of the electrochemical process; however, the possibility of other volatile products cannot be dismissed. Addition of solid benzoquinone to acetate media is followed by slow formation of a brownish black color that is concluded to result from one or more humic compounds produced by condensation of benzoquinone; however, it cannot be concluded whether the condensation reaction is a necessary prerequisite to successful electrochemical incineration of benzoquinone. Optimal operating conditions suggested for electrochemical incineration of benzoquinone in acetate buffer include heating of the anode (e.g., 60 C) to increase the rate of anodic discharge of H{sub 2}O and, thereby, decrease the anodic overpotential. Evidence also is presented that the rate of electrochemical incineration is enhanced slightly by the addition of a traces of Fe(III) with reduction of dioxygen (O{sub 2}) to hydrogen peroxide (H{sub 2}O{sub 2}) at a cooled stainless steel cathodemore » (e.g., 15 C) in an undivided cell.« less

Selective sequential dissolution techniques for trace metals in arid‐zone soils: The carbonate dissolution step

Abstract: Dissolution capacity and kinetics of carbonates by sodium acetate (NaOAc)‐acetic acid (HOAc) at various pHs were studied. A comparative study of the selectivity, specificity, and effectivity of NaOAc‐HOAc solution on carbonate bound fraction during the sequential selective dissolution procedure was conducted by comparing the dissolution of major and trace elements from arid zone soils by this buffer solution at various pHs. The effect of the pH of NaOAc‐HOAc solution on the following fractions in the sequential selective dissolution procedure was also studied. NaOAc‐HOAc solution at pH 5.5 at a soil to solution ratio of 1:25, can dissolve all the carbonate from calcareous soils with 10–20% of carbonate; at pH 5.0 it can dissolve all the carbonate in soils with about 30–50% calcium carbonate (CaCO3). A second extraction with fresh buffer solution at pH 5.0 is required for soils with more than 50% of carbonate. Six hours of extraction time is generally sufficient for complete carbonate dissolution....

A polypyrrole/three-enzyme electrode for creatinine detection

Abstract: To detect creatinine, an enzyme electrode was made by co-immobilization of three enzymes, creatininase, creatinase, and sarcosine oxidase, in an active polypyrrole (PPy) matrix. Besides platinum, polypyrrole doped with a sulfated phenoxy resin (S-PHE) was used as a base electrode. The device was fabricated by electropolymer-ization of pyrrole in the presence of water soluble polyanions, enzymes, and a phosphate buffer, using the above-mentioned base electrodes. At a potential of 400 mV vs Ag/AgCl, the sensors responded to creatinine with a sufficient sensitivity. Under a nitrogen atmosphere, the response current was higher when PPy/S-PHE was used as a base electrode. As a main transduction pathway, a direct electron transfer from sarcosine oxidase to PPy chains is discussed. In addition, the influences of the different polyanionic dopants, the thickness of the active layer, and the concentration of sarcosine oxidase in preparation solutions on the sensor's performance were examined.

Square-Wave Voltammetric Determination of Uric Acid by Catalytic Oxidation at a Perfluorosulfonated Ionomer/Ruthenium Oxide Pyrochlore Chemically Modified Electrode

Abstract: A Nafion/ruthenium oxide pyrochlore (Pb 2 Ru 2-x Pb x O 7-y ) chemically modified electrode (CME) for the determination of uric acid by Osteryoung square-wave voltammetry (OSWV) is described. Compared to the glassy carbon electrode, the CME exhibits a 150 mV shit of the oxidation potential of uric acid in the cathodic direction and a marked enhancement of the current response. The best instrumental settings for OSWV are 50 Hz modulation frequency, 20 mV modulation amplitude, and 2 mV step height. Linear calibration curves are obtained over the 7.5×10 -5 -5×10 -7 M range in pH 1 buffer solution. The detection limit (3) is 1.1×10 -7 M. Ascorbic acid in less than 10-fold excess does not interfere. The practical analytical utility is illustrated by selective measurements of uric acid in human urine and serum without any preliminary treatmen

A Fiber-Optic Carbon Dioxide Sensor for Fermentation Monitoring

Abstract: We have developed a fiber-optic chemical sensor for determining dissolved carbon dioxide and assessed its performance for the on-line monitoring of fermentation. The sensor operates on the Severinghaus pCO2 electrode principle; it consists of a pH sensitive dye (hydroxypyrenetrisulfonic acid, HPTS) in an HCO3− buffer solution entrapped in an expanded PTFE support held at the distal end of an optical fiber by a gas permeable membrane. CO2 crossing the membrane produces a pH change in the indicator solution. This change is related to the external CO2 concentration by the Henderson-Hasselbach equation. The sensor has a reversible working dissolved CO2 dynamic range of 0–0.25 atm. The sensor can be auto-claved without affecting its calibration. Results are presented for the on-line determination of CO2 production in beer fermentation.

Residence time effects on monoclonal antibody binding to adsorbed fibrinogen

Abstract: Fibrinogen adsorbed to polymeric surfaces and then allowed to reside on the surface while it is kept in a buffer solution for a period of time (the 'residence time') undergoes postadsorptive changes that decrease its SDS elutability, displaceability by plasma, polyclonal antifibrinogen binding, and ability to support platelet adhesion (summarized in Chinn et al. J. Biomed. Mater. Res. 26, 757 (1992)). In order to better understand the nature of the changes in adsorbed fibrinogen, the binding of ten different monoclonal antifibrinogen molecules to fibrinogen adsorbed from plasma to Biomer and several other surfaces has been measured after increasing residence time in buffer. Three of the monoclonal antibodies used bind to sequences that have been implicated in platelet binding to fibrinogen. One of these (M1) binds to the C-terminal region of the gamma chain (402-411), another (R1) binds to the N-terminal region of the A alpha chain containing an RGDF sequence (95-98), and the third (R2) binds to the C-ter...

Proton flux measurements from tissues in buffered solution

Abstract: Proton movement across plant cell membranes is part of many important physiological processes. The net proton flux to or from tissues can be determined non-invasively by measuring the proton electrochemical potential gradient in the adjacent solution. In buffered solution, some of the protons crossing the tissue boundary diffuse as proto-nated buffer whose flux is not included in the flux calculated from the proton (hydrogen ion) electrochemical gradient. In this theoretical paper, it is shown how experimenters can calculate the protonated buffer flux from the measured proton flux in solution. The ratio of these two components of total proton flux depends on the pH of the solution and on the concentration and pK of the buffer. For a given concentration of a buffer which has a single pK, the flux ratio rises with pH when the solution pH is lower than the buffer pK. The slope is about 2 on a log10 scale. As the pH increases above the pK, the flux ratio levels off to approach its maximum. With mixed buffers, or one having two or more pK values, the flux ratios are additive: each buffer acts independently based on its concentration and its pK value. Unbuffered solutions always have the buffering effects of water itself and also of carbonates due to carbon dioxide dissolved from the atmosphere. In unbuffered solutions at pH 6, the flux carried by water and carbonate is about 1 % of the measured proton flux. This validates measurements of proton flux from tissues, made by a number of workers, in unbuffered solutions below pH 6.

Spectroscopic study on the photoreduction of hypocrellin A: generation of semiquinone radical anion and hydroquinone

Abstract: Hypocrellin A (HA) is an efficient phototherapeutic agent. Illumination of HA in dimethylsulphoxide (DMSO) or DMSO-buffer (1:1 by volume, pH > 6.7) generated a strong electron paramagnetic resonance (EPR) signal. This EPR signal was intensified in the presence of reductants. The EPR spectrum obtained was assigned to the semiquinone radical anion of HA (HA .− ) based on a series of experimental results. Decay of HA .− , attributed to a radical-radical reaction, follows second-order kinetics. In acidic DMSO-buffer (1:1 by volume) solution, no EPR signal of HA .− was detected in the absence or presence of reductant. This was explained by a fast disproportionation of radicals, facilitated by protonation of radicals. The spectrophotometric measurements indicated that on illumination HA was directly reduced to its two-electron reduction product, i.e. hydroquinone, in acidic solution. The absorption maximum of the hydroquinone of HA which is at 496 nm at pH 5.8 shifts bathochromically with increase in pH of the medium. In DMSO or DMSO-buffer (1:1 by volume, pH > 6.7) solutions the semiquinone radical anion of HA was also observed spectrophotometrically. The absorption maximum of HA .− is at around 628 nm. Strong intramolecular hydrogen bonding was considered to exist in the chromophore of HA .− .

Identification of the Positional Isomers of 2-Fluorobenzoic acid 1-O-Acyl Glucuronide by Directly Coupled HPLC-NMR

Abstract: Directly coupled HPLC- 1 H NMR was used in the stop-flow mode to separate and rapidly identify an equilibrated mixture of ester glucuronide isomers formed spontaneously by intramolecular rearrangement reactions (internal acyl migration and mutarotation) of 2-fluorobenzoic acid β-1-glucuronide (1-O-(2-fluorobenzoyl)-D-glucopyranuronic acid). The equilibrated mixture of isomers was obtained by incubation of the synthetic 2-fluorobenzoic acid glucuronide in buffer solution (pH 7.4) at 25 °C for 24 h. The β-anomer of the 1-O-acyl glucuronide, and the 2-, 3-, and 4-positional glucuronide isomers (all three as both α- and β-anomers) present in the equilibrium mixture, were all characterized after separation in an isocratic chromatographic system containing phosphate buffer at pH 7.4 and 1% acetonitrile in the mobile phase. The HPLC-NMR investigations also elucidated the mutarotation of the positional glucuronide isomers as well as showing the benefits of the HPLC-NMR technique as a primary analytical tool. This HPLC-NMR method will be of particular value in studies on the acyl migration reactions of nonsteroidal antiinflammatory drug glucuronides which may be related to their toxicological properties.