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PubMed1996

Use of pH 9.5 Tris-HCl buffer containing 5% urea for antigen retrieval immunohistochemistry.

Successful antigen retrieval (AR) immunohistochemistry is dependent on the temperature, heating time, and pH value of the AR solutions. There is no single standardized AR solution, however, that is suitable for all antibodies "routinely" used in surgical pathology for immunostaining archival tissue sections. We tested a variety of AR solutions varying in pH value, chemical composition, and molarity. Based upon preliminary results, we compared three AR solutions: 0.1 M Tris-HCl buffer, pH 9.5, containing 5% urea, 0.1 M Tris-HCl buffer pH 9.5 without urea, and citrate buffer, pH 6.0. Each AR solution was tested with a panel of 34 antibodies using microwave heating for antigen retrieval. The heating conditions were standardized at 10 min and an automated stainer was used to standardize the immunostaining method. The Tris-HCl containing urea was superior to pH 6.0 citrate buffer for 22 antibodies. In 12 cases, Tris-HCl with urea was also superior to Tris-HCl alone. In 12 cases, the intensity was similar for all three retrieval solutions. The staining obtained with Tris-HCl with urea was equal to or better than with pH 6.0 citrate buffer in all cases. The Tris-HCl with urea solution is satisfactory for AR of most antibodies employed in routine surgical pathology.

Antigens↗

PubMed1992

Inactivation of gamma-aminobutyric acid aminotransferase by various amine buffers.

It is hypothesized that buffers capable of forming a Schiff base with the PLP of gamma-aminobutyric acid aminotransferase (GABA-AT) may lead to denaturation and inactivation of the enzyme. On the basis of this hypothesis three new methods for the selective destruction of GABA-AT in GABAse (a commercial bacterial source of a mixture of GABA-AT and succinic semialdehyde dehydrogenase [SSDH]) and from pig brain are described: (1) dialysis against a primary or secondary amine buffer; (2) gel filtration with a primary or secondary amine buffer as eluent; (3) inactivation with gabaculine followed by dialysis or gel filtration with pyrophosphate buffer. The SSDH activity in GABAse, which remains unchanged by all of these methods, may then be used in a coupled assay to measure the activity of GABA-AT from different sources. These results also suggest that the use of primary and secondary amine buffers should be avoided when inhibitors are being tested with GABA-AT.

4-Aminobutyrate Transaminase↗

PubMed1983

Influence of buffering early lactation rations with sodium bicarbonate and magnesium oxide and subsequent withdrawal or addition effects.

Eighty animals (16 first lactation) were assigned alternately at calving to one of four treatments: A) corn silage, B) corn silage + 1.5% sodium bicarbonate and .5% magnesium oxide in the grain mix, C) 50% hay crop silage and 50% corn silage, and D) 50% hay crop silage and corn silage plus 1.5% sodium bicarbonate and .5% magnesium oxide. All rations contained 50% forage and 50% concentrate (dry) fed as a total mixed ration. During the first 8 wk of lactation no differences were detected in mean performance or in weekly patterns for forage programs alone or buffer treatments alone on average daily intake of dry matter, body weight loss, milk yield, or composition. Addition of buffers to hay crop silage and corn silage rations resulted in a milk yield profile with a smaller increase beyond wk 3 postpartum. Cows fed all corn silage rations yielded more milk on the average than cows on hay crop silage and corn silage, regardless of buffer treatments. From wk 9 through 12 of lactation, buffers either were added or withdrawn. Addition or withdrawal of buffers did not alter significantly patterns of milk yield or composition.

Animals↗

PubMed2001

Nitrogen and phosphorus variation in shallow groundwater and assimilation in plants in complex riparian buffer zones.

The study of purification efficiency and nutrient assimilation in plants was made in two riparian buffer zones with a complex of wet meadow and grey alder (Alnus incana) stand. In the less polluted Porijõgi test site, the 31 m wide buffer zone removed 40% of total nitrogen (total-N) and 78% of total phosphorus (total-P), while a heavily polluted 51 m wide buffer zone in Viiratsi retained 85% of total-N and 84% of total-P. The input of nutrients and purification efficiency displayed a significant relationship. The total-N removal in buffer zone was negative when the input value was less than 0.3 mg l(-1) and the purification efficiency was always positive when the input value exceeded 5 mg l(-1). The purification efficiency of total-P was positive when the input value exceeded 0.15 mg l(-1). Grass vegetation plays an important role in nutrient retention in riparian buffer strips. The maximum phytomass production was measured in Porijõgi site where production of the Filipendula ulmaria community was up to 2,358 g m(-2), assimilation of N 32.1 and of P 4.9 g m(-2), respectively. This is much higher than the biomass production and N and P uptake of the grey alders (Alnus incana) at the same site--1,730, 20.5 and 1.5 g m(-2), respectively.

Biomass↗

PubMed2002

Hydrogeological constraints on riparian buffers for reduction of diffuse pollution: examples from the Bear Creek watershed in Iowa, USA.

Riparian Management Systems (RiMS) have been proposed to minimize the impacts of agricultural production and improve water quality in Iowa in the Midwestern USA. As part of RiMS, multispecies riparian buffers have been shown to decrease nutrient, pesticide, and sediment concentrations in runoff from adjacent crop fields. However, their effect on nutrients and pesticides moving in groundwater beneath buffers has been discussed only in limited and idealized hydrogeologic settings. Studies in the Bear Creek watershed of central Iowa show the variability inherent in hydrogeologic systems at the watershed scale, some of which may be favorable or unfavorable to future implementation of buffers. Buffers may be optimized by choosing hydrogeologic systems where a shallow groundwater flow system channels water directly through the riparian buffer at velocities that allow for processes such as denitrification to occur.

Biodegradation, Environmental↗

PubMed2002

[Comparison of different lysis buffers in the assessment of the efficiency of single cell polymerase chain reaction for preimplantation genetic diagnosis].

OBJECTIVE: To set up a highly efficient cell lysis system to increase the efficiency of single cell polymerase chain reaction (PCR) for preimplantation genetic diagnosis (PGD). METHODS: First we picked up one single lymphocyte or blastomere and lysed it in alkaline lysis buffer (ALB), proteinase K buffer (PK) and a new proteinase K lysis buffer compounded by ourselves. Then we amplified the 3rd exon of Gene ALD using nested PCR and compared the amplification efficiencies. RESULTS: The amplification efficiencies of the single lymphocyte in ALB, PK and the lysis buffer compounded by ourselves were 90%, 74%, and 87%; those of the single blastomere were 98%, 77%, and 96%. CONCLUSION: ALB and the lysis buffer compounded by ourselves are superior to PK in the lysis of cells in clinical PGD.

Adult↗

PubMed2004

Toward immobilized antibody microarray optimization: print buffer and storage condition comparisons on performance.

Performance issues in protein arrays differ substantially from DNA microarrays because of the different intrinsic construction and stability of proteins versus DNA, and the impact on biological recognition phenomena required for assay from a surface-immobilized state. Maintenance of the capture bioactivity and performance of antibodies on microarray substrates for relatively long periods (under storage or shipping) requires carefully designed and optimized print buffer and storage conditions. We have focused on selection of several additives added to antibody print buffers with different concentrations, and storage of printed antibody slides under different temperatures from 1 day to 4 weeks. Activities of these slides were compared in capture assay formats in order to compare the effects of print buffer and storage conditions of antibody microarrays. Commercial polymer-coated Optarray slides exhibited better performance over Codelink slides in terms of activity of anti-human TNF alpha and IL-1 beta. IL-1 beta antibodies immobilized on both commercial surfaces showed much higher activity than TNF alpha antibodies, regardless of source or vendor. Addition of 1% (v/v) PEO to the print buffer increased the printed analyte capture activity of IL-1 beta on Optarray slides compared to standard print buffer without any additives. This helps to maintain antibody immobilized analyte capture activity for 4 weeks when stored at -70 degrees C.

Adsorption↗

PubMed2004

DNA amplification fingerprinting using 10 x polymerase chain reaction buffer with ammonium sulfate for human identification.

OBJECTIVE: The polymerase chain reaction (PCR) - based DNA amplification fingerprinting (DAF) or randomly amplified polymorphic DNA (RAPD) is based on a strategy using a single arbitrary oligonucleotide primer to generate anonymous amplification of genomic DNA. On this basic strategy, in this study, we aimed to test individual differences and usefulness of 2 basic primers (5'-CGCGCCGG-3' and 5'-TGCCGAGCTG-3') and examined whether there is a positive effect on results of 10 x PCR buffer with ammonium sulfate. METHODS: A new approach in DNA fingerprinting, 10 x PCR buffer with ammonium sulfate, is presented in the study. Primers with single 8 and 10 nucleotides in length and 2 different PCR buffers with or without ammonium sulfate were used to identify 135 volunteers with no blood relationship. This study was carried out at the Pharmacology Laboratory, University of Gaziantep, School of Medicine, Turkey between 1999 and 2000. RESULTS: An average of 10 major bands representing 500-1500 base pair (bp) in length was determined as amplified DNA products on standard agarose gels for these volunteers. The use of ammonium sulfate in 10 x PCR buffers has increased to 92% success ratio of individual difference obtained from the 8 nucleotides primer. CONCLUSION: With this study, more reliable results can be obtained by using ammonium sulfate in 10 x PCR buffers.

Ammonium Sulfate↗

PubMed2005

Buffer zones as a sink for sediment and phosphorus between the field and stream: Danish field experiences.

Rill erosion, surface runoff and storage of sediment and phosphorus in buffer zones were investigated during three winters on ca. 140 arable field slope units in twenty places in Denmark covering all landscape types, climate gradients and dominant soil types. The dominant soils are Alfisols and Spodosols, with textural composition typically ranging from sand to loam. The average slope is 7% (range: 2-20%) and median buffer zone width was 8.3 m (range: 0.6-125 m). The geometric mean annual rill erosion was 0.33 m3 ha(-1) equalling to 495 kg sediment ha(-1) and 0.25 kg P ha(-1). The deposition of sediment on the field, in the buffer zone and delivery of soil to the stream was surveyed within the 140 slope units following three winters. Deposition of sediment in the buffer zone was observed in 31% (1997/98), 31% (1998/99) and 29% (1999/2000) of the slope units. Delivery of soil across the edge of the stream was observed in 23% (1997/98), 17% (1998/99) and 25% (1999/2000) of the 140 slope units. Median dissolved P concentration in surface runoff was 0.18 mg Pl(-1) (range: 0.029-16.294 mg Pl(-1)). A probability model was developed to predict the efficiency of different widths of buffer zones.

Agriculture↗

PubMed1986

Effects of permeant buffers on the initiation of photosynchronous phosphorylation and postillumination phosphorylation in chloroplasts.

Under canonical chemiosmotic formulations, the development of a delocalized transmembrane proton gradient should precede and, in the absence of a membrane potential, should account for all the capacity of an energy transducing system to synthesize ATP. Furthermore, any agents, such as permeant proton-absorbing buffers, that slow down the kinetics of the development of this gradient should, consequently, delay ATP synthesis. We have studied the very early (0 through 1000 ms) steps of photosynthetic ATP synthesis utilizing real-time, rapid flow-quench techniques. We have investigated the effect(s) that permeant buffers exert on this process where these buffers show no uncoupling effects, and the transmembrane potential has been collapsed by valinomycin and K+. Experimentally this system was dissected into two ATP synthesizing components, as follows: synthesis of ATP strictly concomitant with light influx and unaffected by the addition of permeant buffers. We refer to this as photosynchronous phosphorylation and synthesis of ATP monitored after the light was extinguished and which was greatly diminished by the addition of proton-absorbing permeant buffers, thus exhibiting the characteristics of conventional postillumination phosphorylation, and we suggest that it represents part of capacitance phosphorylation. The potential for capacitance phosphorylation initiates very rapidly under light and gradually builds up to steady-state level, and it is governed by canonical chemiosmotic principles. We estimate that its contribution to overall ATP yield is minimal during the first few cycles of the system and that it increases gradually towards steady state when it contributes to the majority of ATP synthesized. Neither a delocalized transmembrane proton gradient nor a strictly localized intramembrane proton pathway can account for these observations so we have proposed that a gating mechanism exists which delivers intramembrane protons initially directly to the ATP synthetase complex but subsequently to the lumen as well, and thus, allows the lumen to act as a capacitor during the steady state. This study can reconcile the findings of Ort et al. (Ort, D. R., Dilley, R. A., and Good, N. E. (1976) Biochim. Biophys. Acta 449, 108-124) with the contrasting findings of Vinkler et al. (Vinkler, C., Avron, M., and Boyer, P. D. (1980) J. Biol. Chem. 255, 2263-2266) through the opposite effects which osmotic strength and KCl concentration exert on the two ATP synthetic phases (during and after illumination) of the rapid flash technique used in those studies.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Triphosphate↗

PubMed1983

Secretion rate and buffer effect of resting and stimulated whole saliva as a function of age and sex.

Duplicate determinations of secretion rate and buffer effect of resting and stimulated whole saliva were made in 629 adults (286 males and 343 females) within an interval of 1-2 weeks. The subjects were arbitrarily classified into four separate age-groups. For all variables studied, a highly significant correlation was obtained between the duplicate tests. The secretion rate of resting as well as stimulated saliva was significantly lower for females than for males. For females, the resting secretion rate was negatively correlated with age. The buffer effect was also significantly lower in the females for both resting and stimulated saliva. For the females, the buffer effect was positively correlated with age and therefore, with advancing age, the females tended to catch up with the males. Irrespective of sex, the secretion rate of resting saliva was highly correlated to that of stimulated saliva. This relationship was established also for the buffer effect. However, between secretion rate and buffer effect a correlation was observed only for stimulated saliva.

Adolescent↗

PubMed1994

Evaluation of highly buffered low-calcium solution for long-term preservation of the heart. Comparison with University of Wisconsin solution.

Myocardial preservation for prolonged ischemia has traditionally centered around deep hypothermia with metabolic arrest. This approach is limited in tolerable ischemic time by the state of energy reserves at the onset of ischemia, because anaerobic glycolysis during ischemia is limited by end-product accumulation (lactate, alanine, and H+). In this study we evaluated a novel preservation solution containing the basic amino acid histidine to buffer H+, glucose as substrate, and low sodium and calcium concentrations to mimic the intracellular ionic environment. Isolated rabbit hearts were subjected to hypothermic ischemia for 8 and 16 hours at 4 degrees and 21 degrees C followed by reperfusion. The buffered solution was compared to University of Wisconsin solution (high potassium). Intracellular pH was maintained at preischemic levels in the buffered solution hearts at 21 degrees C, and this was associated with better preservation of high energy stores and recovery of contractile function. Developed pressure recovered to 90% +/- 3% of preischemic values after 16 hours of 21 degrees C ischemia with the buffered solution as compared with 79% +/- 2% in the University of Wisconsin group at 4 degrees C (contracture occurred in the University of Wisconsin hearts at 21 degrees C). The optimal temperature in the buffered solution hearts was 13 degrees C, and with this temperature acceptable recovery of contractile function was seen after 24 hours of ischemia. On the basis of these results, we conclude that promoting anaerobic glycolysis during ischemia achieves superior prolonged preservation of energetic and contractile function of the heart.

Animals↗

PubMedDate not supplied

The apparent buffer value of cerebrospinal fluid in acute hypercapnia.

Buffer mechanism of cerebrospinal fluid (CSF) against acute hypercapnia was studied in eighteen dogs. The dynamic response of CSF to a stepwise change of CO2 concentration in inspired gas (room air -- 6% CO2 -- 12% CO2) was observed in eleven dogs, maintaining each condition for two hours. The changes in CSF acidity were less than that in arterial blood, while increases of bicarbonate ion concentration [HCO3-] in CSF were more prominent. Apparent buffer values, delta[HCO3-]/deltapH, were calculated from the results in different levels of CO2 breathing : they were 22.7 slykes from room air to 6% CO2 (step 1), and 39.7 slykes from 6% to 12% CO2 (step 2). Similar experiments were performed in seven dogs, suppressing carbonic anhydrase activity by systemic administration of acetazolamide. Apparent buffer values of CSF were 14.4 slykes in step 1 and 16.0 slykes in step 2. From the result we conclude : 1) that the activity of buffer mechanism of CSF in respiratory acidosis is PCO2 dependent and becomes stronger when PCO2 of CSF increases ; 2) for the explanation of this characteristic buffer mechanism of CSF, participation of carbonic anhydrase is suggested for transport mechanism of bicarbonate ion into CSF.

Acetazolamide↗

PubMed1997

[Optimum choice for the cultivation of CFU-GM in media buffered with HEPES].

The effects of media buffered with HEPES of different concentrations and different incubation periods on the proliferation and differentiation of CFU-GM were studied under the standard condition for culture. The results exhibited that when the incubation period prolonged, the pH values of culture system raised and the colony formation of CFU-GM decreased; the culture system added with proper HEPES buffer solution could maintain and increase the colony formation of CFU-GM, especially the culture system in which the pH value was not adjusted before the experiment. We also found that media buffered with HEPES could change the ratio of different colony types. This showed that media buffered with HEPES could not only stimulate the proliferation of CFU-GM, but also affect its differentiation. The results suggest that media buffered with HEPES are better than those without HEPES for the culture of hematopoietic progenitor cells, and the optimum concentration is 10-20 mmol.L-1.

Animals↗

PubMed1979

Human tear buffering capacity.

With the use of a closed chamber microelectrode system, we measured the relative buffering capacities of 490 human tear samples from young healthy adults. The buffering capacities of the 457 waking-hour samples did show small but regular oscillations that were similar to those previously reported for blood and tear pH, but only rarely did the buffering capacity of one sample approach double that of another. The buffering capacities of 33 tear samples, associated with periods of prolonged eye closure (sleep), were not significantly different (P less than 0.5) from those of the open eye.

Adult↗

PubMed1991

Immunogenicity of tetravalent rhesus rotavirus vaccine administered with buffer and oral polio vaccine.

Between January and November 1989, we studied 174 infants aged 6 to 16 weeks in a randomized clinical trial to (1) determine the immunogenicity of a single dose of tetravalent rhesus rotavirus vaccine (RRV-TV) when administered with three different buffer regimens: no antacid buffer and small-volume (2.5-mL) and large-volume (30-mL) antacid buffer; and (2) examine the potential interference of RRV-TV on the immune response to oral polio vaccine. Immunogenicity of RRV-TV, measured as a fourfold rise in antibody titers to rotavirus, was similar in the groups receiving small- and large-dose buffer (45% and 49%, respectively) and significantly less in the group that received RRV-TV alone (23%). Administration of RRV-TV with oral polio vaccine did not significantly interfere with the neutralization response of oral polio vaccine poliovirus serotypes 1, 2, or 3, and overall, 29%, 87%, and 24% of the infants had a fourfold rise in titer to each serotype, respectively.

Antibodies, Viral↗

PubMed1997

Effects of buffering intracellular free calcium on neutrophil migration through three-dimensional matrices.

Repeated transient increases in intracellular free calcium levels ([Ca2-]i) are required for polymorphonuclear neutrophil migration on two-dimensional surfaces coated with fibronectin or vitronectin. Cells in which [Ca2+]i is buffered with quin2 become stuck on these substrates. Neutrophils migrating through the extracellular matrix in vivo encounter these and other substrates in a three-dimensional architecture that may alter the spatial distribution of adhesion receptors in contact with the matrix. In this study, we used fluorescence confocal microscopy to obtain moving three-dimensional images of neutrophils migrating through a biological tissue (human amnion) in the presence and absence of [Ca2+]i-buffering with quin2. In the absence of buffering, [Ca2+]i transients similar to those seen in cells migrating in two-dimensions were observed. [Ca2+]i-buffered neutrophils were able to migrate into the matrix, but they became attached firmly to the substrate at the rear of the cell, resulting in a drastically elongated morphology. Immunofluorescence revealed that neutrophils adhered to regions of the matrix that contained fibronectin. RGD-containing peptides and antibodies that block integrin adhesion receptors for fibronectin and vitronectin were able to rescue the migration of quin2-treated cells through three-dimensional gels containing fibronectin and vitronectin. These data show that neutrophils migrating through physiologically relevant, three-dimensional matrices undergo repetitive increases in [Ca2+]i that are required for integrin-mediated detachment from the matrix.

Amnion↗

PubMed2000

Single-strand conformation polymorphism analysis by capillary zone electrophoresis in neutral pH buffer.

Sensitivity of single-strand conformation polymorphism (SSCP) analysis of polymerase chain reaction (PCR) products was reported to be lower in capillary zone electrophoresis (CZE) compared to conventional slab gel electrophoresis. We examined the effects of buffer ion type, pH, and temperature in an attempt to improve the mutation detectability in the SSCP-CZE mode. It was noted that, by utilizing short-chain polyacrylamide as sieving media while simultaneously lowering the temperature, there was no improvement of conformer detectability. On the contrary, there was a large increment in conformers' resolution by running samples in a lower-pH buffer system. The effects of different buffering ions and pH values were investigated. By using a new buffer system, consisting of 35 mM 2-(N-morpholino)propanesulfonic acid (MES), 30 mM tris(hydroxymethyl)aminomethane (Tris), 1 mM ethylene diaminetetraacetic acid (EDTA), pH 6.8, and keeping constant all the other conditions, such as temperature, sieving, applied voltage, capillary length, and inner diameter (ID), there was a remarkable improvement in resolution and the sensitivity became comparable to that of slab gel systems.

Buffers↗