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Fertility and Sterility Sep 1992
Review
Topics: Buffers; Egg Yolk; Female; Fertilization in Vitro; Humans; Male; Sperm-Ovum Interactions; Spermatozoa; Tromethamine
PubMed: 1521639
DOI: 10.1016/s0015-0282(16)55248-0 -
Nature Protocols Mar 2020It is important to assess the identity and purity of proteins and protein complexes during and after protein purification to ensure that samples are of sufficient...
It is important to assess the identity and purity of proteins and protein complexes during and after protein purification to ensure that samples are of sufficient quality for further biochemical and structural characterization, as well as for use in consumer products, chemical processes and therapeutics. Native mass spectrometry (nMS) has become an important tool in protein analysis due to its ability to retain non-covalent interactions during measurements, making it possible to obtain protein structural information with high sensitivity and at high speed. Interferences from the presence of non-volatiles are typically alleviated by offline buffer exchange, which is time-consuming and difficult to automate. We provide a protocol for rapid online buffer exchange (OBE) nMS to directly screen structural features of pre-purified proteins, protein complexes or clarified cell lysates. In the liquid chromatography coupled to mass spectrometry (LC-MS) approach described in this protocol, samples in MS-incompatible conditions are injected onto a short size-exclusion chromatography column. Proteins and protein complexes are separated from small molecule non-volatile buffer components using an aqueous, non-denaturing mobile phase. Eluted proteins and protein complexes are detected by the mass spectrometer after electrospray ionization. Mass spectra can inform regarding protein sample purity and oligomerization, and additional tandem mass spectra can help to further obtain information on protein complex subunits. Information obtained by OBE nMS can be used for fast (<5 min) quality control and can further guide protein expression and purification optimization.
Topics: Buffers; Chemistry Techniques, Analytical; Chromatography, Gel; Mass Spectrometry; Proteins
PubMed: 32005983
DOI: 10.1038/s41596-019-0281-0 -
Bulletin of Mathematical Biology Dec 2022The existence and properties of intracellular waves of increased free cytoplasmic calcium concentration (calcium waves) are strongly affected by the binding and...
The existence and properties of intracellular waves of increased free cytoplasmic calcium concentration (calcium waves) are strongly affected by the binding and unbinding of calcium ions to a multitude of different buffers in the cell. These buffers can be mobile or immobile and, in general, have multiple binding sites that are not independent. Previous theoretical studies have focused on the case when each buffer molecule binds a single calcium ion. In this study, we analyze how calcium waves are affected by calcium buffers with two non-independent binding sites, and show that the interactions between the calcium binding sites can result in the emergence of new behaviors. In particular, for certain combinations of kinetic parameters, the profiles of buffer molecules with one calcium ion bound can be non-monotone.
Topics: Calcium Signaling; Calcium; Buffers; Models, Biological; Mathematical Concepts; Binding Sites
PubMed: 36585964
DOI: 10.1007/s11538-022-01109-0 -
Biosensors Nov 2021We investigated the stability of silver nanoisland films, which were formed on glass surface by the method of out-diffusion, in biocompatible buffers and the...
We investigated the stability of silver nanoisland films, which were formed on glass surface by the method of out-diffusion, in biocompatible buffers and the applicability of the films in surface enhanced Raman scattering (SERS). We have shown that silver nanoisland films are stable in one of the most widespread in biological studies buffer-phosphate buffer saline (PBS), and in 1:100 water-diluted PBS, in the PBS-based buffer, in which NaCl is replaced by the same amount of NaClO, and in acidic phosphate buffer. At the same time, the replacement of NaCl in PBS by N(CH)Cl leads to the degradation of the nanoislands. It was shown that after exposure to PBS the nanoisland films provided a good SERS signal from a monolayer of 1,2-di(4-pyridyl)ethylene (BPE), which makes silver nanoisland films promising for biosensor applications. Additionally, in our experiments, we registered for the first time that silver nanoparticles formed in the bulk of the samples dissolved after exposing to PBS, while nanoislands on the glass surface stayed unchanged. We associate this phenomenon with the interaction of ions contained in PBS solution with silver, which results in the shift of corresponding chemical equilibrium.
Topics: Biocompatible Materials; Buffers; Metal Nanoparticles; Phosphates; Silver; Sodium Chloride; Spectrum Analysis, Raman
PubMed: 34821664
DOI: 10.3390/bios11110448 -
International Journal of Pharmaceutics Feb 2013Oxytocin is a peptide drug used to induce labor and prevent bleeding after childbirth. Due to its instability, transport and storage of oxytocin formulations under...
Oxytocin is a peptide drug used to induce labor and prevent bleeding after childbirth. Due to its instability, transport and storage of oxytocin formulations under tropical conditions is problematic. In a previous study, we have found that the stability of oxytocin in aspartate buffered formulation is improved by the addition of divalent metal ions (unpublished results). The stabilizing effect of Zn(2+) was by far superior compared to that of Mg(2+). In addition, it was found that stabilization correlated well with the ability of the divalent metal ions to interact with oxytocin in aspartate buffer. Furthermore, LC-MS (MS) measurements indicated that the combination of aspartate buffer and Zn(2+) in particular suppressed intermolecular degradation reactions near the Cys(1,6) disulfide bridge. These results lead to the hypothesis that in aspartate buffer, Zn(2+) changes the conformation of oxytocin in such a way that the Cys(1,6) disulfide bridge is shielded from its environment thereby suppressing intermolecular reactions involving this region of the molecule. To verify this hypothesis, we investigate here the conformation of oxytocin in aspartate buffer in the presence of Mg(2+) or Zn(2+), using 2D NOESY, TOCSY, (1)H-(13)C HSQC and (1)H-(15)N HSQC NMR spectroscopy. Almost all (1)H, (13)C and (15)N resonances of oxytocin could be assigned using HSQC spectroscopy, without the need for (13)C or (15)N enrichment. (1)H-(13)C and (1)H-(15)N HSQC spectra showed that aspartate buffer alone induces minor changes in oxytocin in D2O, with the largest chemical shift changes observed for Cys(1). Zn(2+) causes more extensive changes in oxytocin in aqueous solution than Mg(2+). Our findings suggest that the carboxylate group of aspartate neutralizes the positive charge of the N-terminus of Cys(1), allowing the interactions with Zn(2+) to become more favorable. These interactions may explain the protection of the disulfide bridge against intermolecular reactions that lead to dimerization.
Topics: Aspartic Acid; Buffers; Drug Stability; Magnesium; Magnetic Resonance Spectroscopy; Oxytocin; Protein Conformation; Zinc
PubMed: 23376504
DOI: 10.1016/j.ijpharm.2013.01.051 -
Methods in Molecular Biology (Clifton,... 2014Cell transfection efficiency often determines the success of cell-based gene therapy. Cell transfection via Nucleofector technology yields high transfection efficiency...
Cell transfection efficiency often determines the success of cell-based gene therapy. Cell transfection via Nucleofector technology yields high transfection efficiency and low cytotoxicity. However, owing to trade secrecy, the components in each buffer are unknown, which not only increases the cost of electroporation studies but also limits the application of Nucleofector in clinical cell-based gene therapies. Thus, we developed a three-step method to determine the optimal conditions, including buffer, program, and additional polymer, in electroporation for multiple cancers and stem cell lines. This method could reduce the cost, allow researchers to find the optimal electroporation conditions for their cell lines of interest, and greatly boost the application potential of electroporation in clinical cell-based gene therapies.
Topics: Animals; Buffers; Cell Line; Cell- and Tissue-Based Therapy; Electroporation; Humans; Polymers; Transfection
PubMed: 24510811
DOI: 10.1007/978-1-4614-9632-8_4 -
Biophysical Journal May 2021We examine closed-form approximations for the equilibrium Ca and buffer concentrations near a point Ca source representing a Ca channel, in the presence of a mobile...
We examine closed-form approximations for the equilibrium Ca and buffer concentrations near a point Ca source representing a Ca channel, in the presence of a mobile buffer with two Ca binding sites activated sequentially and possessing distinct binding affinities and kinetics. This allows us to model the impact on Ca nanodomains of realistic endogenous Ca buffers characterized by cooperative Ca binding, such as calretinin. The approximations we present involve a combination or rational and exponential functions, whose parameters are constrained using the series interpolation method that we recently introduced for the case of simpler Ca buffers with a single Ca binding site. We conduct extensive parameter sensitivity analysis and show that the obtained closed-form approximations achieve reasonable qualitative accuracy for a wide range of buffer's Ca binding properties and other relevant model parameters. In particular, the accuracy of the derived approximants exceeds that of the rapid buffering approximation in large portions of the relevant parameter space.
Topics: Binding Sites; Buffers; Calcium; Kinetics
PubMed: 33771472
DOI: 10.1016/j.bpj.2021.03.015 -
Applied Microbiology and Biotechnology Dec 2006Nucleic acid separation is an increasingly important tool for molecular biology. Before modern technologies could be used, nucleic acid separation had been a time- and... (Review)
Review
Nucleic acid separation is an increasingly important tool for molecular biology. Before modern technologies could be used, nucleic acid separation had been a time- and work-consuming process based on several extraction and centrifugation steps, often limited by small yields and low purities of the separation products, and not suited for automation and up-scaling. During the last few years, specifically functionalised magnetic particles were developed. Together with an appropriate buffer system, they allow for the quick and efficient purification directly after their extraction from crude cell extracts. Centrifugation steps were avoided. In addition, the new approach provided for an easy automation of the entire process and the isolation of nucleic acids from larger sample volumes. This review describes traditional methods and methods based on magnetic particles for nucleic acid purification. The synthesis of a variety of magnetic particles is presented in more detail. Various suppliers of magnetic particles for nucleic acid separation as well as suppliers offering particle-based kits for a variety of different sample materials are listed. Furthermore, commercially available manual magnetic separators and automated systems for magnetic particle handling and liquid handling are mentioned.
Topics: Automation; Buffers; Magnetics; Nucleic Acid Amplification Techniques; Nucleic Acids
PubMed: 17063328
DOI: 10.1007/s00253-006-0675-0 -
Journal of Pharmaceutical Sciences Sep 2015Bicarbonate is the main buffer in the small intestine and it is well known that buffer properties such as pKa can affect the dissolution rate of ionizable drugs.... (Comparative Study)
Comparative Study
Bicarbonate is the main buffer in the small intestine and it is well known that buffer properties such as pKa can affect the dissolution rate of ionizable drugs. However, bicarbonate buffer is complicated to work with experimentally. Finding a suitable substitute for bicarbonate buffer may provide a way to perform more physiologically relevant dissolution tests. The dissolution of weak acid and weak base drugs was conducted in bicarbonate and phosphate buffer using rotating disk dissolution methodology. Experimental results were compared with the predicted results using the film model approach of (Mooney K, Mintun M, Himmelstein K, Stella V. 1981. J Pharm Sci 70(1):22-32) based on equilibrium assumptions as well as a model accounting for the slow hydration reaction, CO2 + H2 O → H2 CO3 . Assuming carbonic acid is irreversible in the dehydration direction: CO2 + H2 O ← H2 CO3 , the transport analysis can accurately predict rotating disk dissolution of weak acid and weak base drugs in bicarbonate buffer. The predictions show that matching the dissolution of weak acid and weak base drugs in phosphate and bicarbonate buffer is possible. The phosphate buffer concentration necessary to match physiologically relevant bicarbonate buffer [e.g., 10.5 mM (HCO3 (-) ), pH = 6.5] is typically in the range of 1-25 mM and is very dependent upon drug solubility and pKa .
Topics: Acid-Base Equilibrium; Acids; Bicarbonates; Buffers; Carbonic Acid; Hydrogen-Ion Concentration; Phosphates; Predictive Value of Tests; Solubility
PubMed: 25980464
DOI: 10.1002/jps.24460 -
Biosensors Aug 2023We present a novel and easy approach using a silicon-based impedance chip to determine the concentration of the given aqueous buffer solution. An accurate determination...
We present a novel and easy approach using a silicon-based impedance chip to determine the concentration of the given aqueous buffer solution. An accurate determination of the post-dilution concentration of the buffers is necessary for ensuring optimal buffer capacity, pH stability, and to assess solution reproducibility. In this study, we focused on phosphate buffer as the test liquid to achieve precise post-dilution concentration determinations. The impedance chip consisting of a top gold ring electrode, where a test volume of 20 μL to 30 μL of phosphate buffer was introduced for impedance measurements within the frequency range of 40 Hz to 1 MHz. For impedance investigation, we used phosphate buffers with three different pH values, and the impedance was measured after diluting the phosphate buffers to a concentration of 1.00 M, 0.75 M, 0.50 M, 0.25 M, 0.10 M, 0.05 M, and 0.01 M. In order to analyze the distinctive changes in the measured impedance, an equivalent circuit was proposed and modeled. From the impedance modeling, we report that the circuit parameter R showed exponential dependence on the concentration of phosphate buffer and no dependence on the pH values of the phosphate buffer and on the added volume inside the ring electrode. The proposed silicon-based impedance chip is quick and uses reduced liquid volume for post-dilution concentration measurements of buffers and has perspective applications in the pharmaceutical and biological domains for regulating, monitoring, and quality control of the buffers.
Topics: Buffers; Hydrogen-Ion Concentration; Silicon; Electric Impedance; Reproducibility of Results; Phosphates
PubMed: 37754075
DOI: 10.3390/bios13090841