Common Reagents Research Articles

Variations on Negative Stain Electron Microscopy Methods: Tools for Tackling Challenging Systems

Negative stain electron microscopy (EM) allows relatively simple and quick observation of macromolecules and macromolecular complexes through the use of contrast enhancing stain reagent. Although limited in resolution to a maximum of ~18 - 20 Å, negative stain EM is useful for a variety of biological problems and also provides a rapid means of assessing samples for cryo-electron microscopy (cryo-EM). The negative stain workflow is straightforward method; the sample is adsorbed onto a substrate, then a stain is applied, blotted, and dried to produce a thin layer of electron dense stain in which the particles are embedded. Individual samples can, however, behave in markedly different ways under varying staining conditions. This has led to the development of a large variety of substrate preparation techniques, negative staining reagents, and grid washing and blotting techniques. Determining the most appropriate technique for each individual sample must be done on a case-by-case basis and a microscopist must have access to a variety of different techniques to achieve the highest-quality negative stain results. Detailed protocols for two different substrate preparation methods and three different blotting techniques are provided, and an example of a sample that shows markedly different results depending on the method used is shown. In addition, the preparation of some common negative staining reagents, and two novel Lanthanide-based stains, is described with discussion regarding the use of each.

Combined effects of antimony and sodium diethyldithiocarbamate on soil microbial activity and speciation change of heavy metals. Implications for contaminated lands hazardous material pollution in nonferrous metal mining areas

The combined effects of antimony (Sb) and sodium diethyldithiocarbamate (DDTC), a common organic flotation reagent, on soil microbial activity and speciation changes of heavy metals were investigated for the first time. The results showed that the exchangeable fraction of Sb was transformed to a stable residual fraction during the incubation period, and the addition of DDTC promoted the transformation compared with single Sb pollution, probably because DDTC can react with heavy metals to form a complex. In addition, the presence of DDTC and Sb inhibited the soil microbial activity to varying degrees. The growth rate constant k of different interaction systems was in the following order on the 28th day: control group ≥ single DDTC pollution > combined pollution > single Sb pollution. A correlation analysis showed that the concentration of exchangeable Sb was the primary factor that affected the toxic reaction under combined pollution conditions, and it significantly affected the characteristics of the soil microorganisms. All the observations provide useful information for a better understanding of the toxic effects and potential risks of combined Sb and DDTC pollution in antimony mining areas.

Common murine immunoglobulin detection reagents have diminished reactivity with IgG3 – A vulnerability to misinterpretation

Methods designed to monitor humoral immune responses, in a variety of settings, typically use a broadly reactive detection reagent (e.g. polyclonal anti-Ig (immunoglobulin)) in order to characterize antibody responses. In the context of murine models of immunity, which are widely used, this would typically be antisera to mouse Ig or mouse IgG. However, there are 4 different subtypes of mouse IgG; thus, the validity of the above approach, as a general screen for humoral immune responses, depends upon the assumption that the antisera recognize all IgG subtypes. This seems like a reasonable assumption, since polyclonal antisera recognize multiple epitopes; however, herein we report that two commercial sources of goat anti-mouse Ig are hyporeactive with IgG3. Given that relative IgG3 levels are different in distinct types of immune response, these findings demonstrate a potential for misinterpretation, and suggest a need to modify immunological methods in this context.

Framework for laboratory harmonization of folate measurements in low- and middle-income countries and regions.

The measurement of serum and red blood cell folate, two commonly used biomarkers of folate status in populations, is complicated by analytical and data interpretation challenges. Folate results show poor comparability across laboratories, even using the same analytical technique. The folate microbiologic assay produces accurate results and requires simple instrumentation. Thus, it could be set up and maintained in low- and middle-income country laboratories. However, the assay has to be harmonized through the use of common critical reagents (e.g., microorganism and folate calibrator) in order to produce comparable results across laboratories and over time, so that the same cutoff values can be applied across surveys. There is a limited need for blood folate measurements in a country owing to the periodic nature of surveys. Having a network of regional resource laboratories proficient in conducting the folate microbiologic assay and willing and able to perform service work for other countries will be the most efficient way to create an infrastructure wherein qualified laboratories produce reliable blood folate data. Continuous participation of these laboratories in a certification program can verify and document their proficiency. If the resource laboratories conduct the work on a fee-for-service basis, they could become self-sustaining in the long run.

Detection of Proteins in Polyacrylamide Gels via Prelabeling by Isatoic Anhydride.

Fluorescent methods for staining of proteins in polyacrylamide gels are obtaining increasing attention due to their advantages including high sensitivity and rapidness. In the present chapter we describe, step by step, a rapid and inexpensive fluorescent prelabeling method based on derivatization of proteins with isatoic anhydride. This method allows researchers to detect less than 2 ng of standard proteins per band in less than 15 min. Although this method is sensitive, inexpensive, rapid, compatible with most of common biological reagents and is able to detect proteins in crude cell extract, covalent binding of isatoic anhydride to protein lysine residues makes it unsuitable for processes in which post-electrophoresis analysis is required. Moreover, isatoic anhydride derivatization induces a small band broadening.

Efficiency estimation of cation-exchange recovery of heavy metals from solutions containing their mixtures

The object of research is the washing waste water of galvanic plants containing heavy metal cations. The most common reagent methods for purification of galvanic drains do not provide the required degree of water purification, are accompanied by the loss of valuable components and the formation of significant amounts of toxic sludge. Ion exchange is promising in development of wastewater treatment of galvanic productions. One of the most important problems of ion-exchange technology is the processes of regeneration of ion exchangers, and, in particular, the recycling of spent regeneration solutions. The most promising in the recovery of regeneration solutions is the electrochemical method. However, electroextraction of zinc and nickel is practically impossible from acidic solutions without separation of the electrode space by the membrane. In addition, effluents from the galvanic industry contain mixtures of heavy metal ions. This complicates the process of wastewater treatment and the return of valuable components to production. In the course of the study, a strongly acidic cation exchanger KU-2-8 in the Na + -form is used to study the processes of combined sorption of heavy metal ions. Regeneration of the cation exchanger is carried out with solutions of sulfuric and hydrochloric acids. Electroextraction of zinc and nickel from acid regeneration solutions is carried out in a two-cell electrolyzer with an anion-exchange membrane MA 41. The obtained results indicate that under conditions of the concentration of heavy metals on cation exchanger KU-2-8, efficient separation can’t be achieved. It is established that ions of heavy metals are sorbed individually and in mixtures with other ions of heavy metals are effectively desorbed by solutions of sulfuric and hydrochloric acids. The use of a two-cell electrolyzer, in contrast to carrying out electrolysis processes without separation of the electrode space, almost completely eliminates zinc and nickel ions from acidic regenerative rods. And to obtain a solution of sulfuric acid in the anode chamber at a concentration of 239–651 meq/dm 3 .

Enrichment of gamma-aminobutyric acid in bean sprouts: Exploring biosynthesis of plant metabolite using common household reagents.

The enrichment of plant foods with gamma-aminobutyric acid (GABA) is currently an interesting issue in the field of nutraceuticals and can be used as an experiment for upper-division undergraduate students. Here, an interdisciplinary hands-on experiment to produce GABA-enriched mung bean sprouts using common household reagents is described. Based on the GABA biosynthesis pathways, two key factors, being the availability of l-glutamic acid and the acidification of the germination environment, were chosen for the study of the effects on the enhancement of GABA levels. The activities not only led students to a deeper understanding of biochemistry contents, but also gave the students the opportunity to work with experimental design, analytical chemistry, and statistical data analysis. Furthermore, since mung bean sprouts are familiar foods and the reagents used for germination are easily obtainable and generally recognized as safe, the optimal protocol investigated in the lab could be further applied to the production of bean sprouts with enhanced nutritional values in everyday life, promoting the transfer of knowledge learned in school to practical environments such as home and community. © 2017 by The International Union of Biochemistry and Molecular Biology, 46(2):155-161, 2018.

CITU: A Peptide and Decarboxylative Coupling Reagent.

Tetrachloro-N-hydroxyphthalimide tetramethyluronium hexafluorophosphate (CITU) is disclosed as a convenient and economical reagent for both acylation and decarboxylative cross-coupling chemistries. Within the former set of reactions, CITU displays reactivity similar to that of common coupling reagents, but with increased safety and reduced cost. Within the latter, increased yields, more rapid conversion, and a simplified procedure are possible across a range of reported decarboxylative transformations.

Use of SF6 for the production of electrophilic 18F-fluorination reagents

Electrophilic 18F-fluorination is an important method for production of tracers for positron emission tomography. The most commonly employed 18F-fluorination reagents [18F]F2, [18F]Selectfluor bis(triflate) and [18F]NFSi allow for the synthetically simple and fast introduction of fluorine-18 into organic molecules, in particular those containing electron rich aromatic rings. For the time being the best method for the production of [18F]F2 gas, the most common electrophilic reagent, is a post-target production which applies a high voltage electrical discharge to promote the 19F/18F isotopic exchange between [18F]MeF and carrier F2 gas.The aim of this study was to develop a new method for the production of electrophilic 18F-fluorination reagents which would avoid use of toxic and very reactive carrier F2 gas. In this study the use of SF6 as an alternative to carrier F2 gas was tested. Experiments were carried out in two different matrix gases: Ne and Xe. The electrophilic 18F-fluorination reagent produced was used for the labelling of two model compounds (6-[18F]fluoro-L-DOPA and [18F]Selectfluor bis(triflate)) in order to prove its feasibility.This proof-of-concept study showed that SF6 in Xe can be used in place of F2 for the production of electrophilic [18F]F2 gas. When SF6 in Ne was used only trace amounts of [18F]F2 was observed.

P−B Desulfurization: An Enabling Method for Protein Chemical Synthesis and Site‐Specific Deuteration

AbstractCysteine‐mediated native chemical ligation is a powerful method for protein chemical synthesis. Herein, we report an unprecedentedly mild system (TCEP/NaBH4 or TCEP/LiBEt3H; TCEP=tris(2‐carboxyethyl)phosphine) for chemoselective peptide desulfurization to achieve effective protein synthesis via the native chemical ligation–desulfurization approach. This method, termed P−B desulfurization, features usage of common reagents, simplicity of operation, robustness, high yields, clean conversion, and versatile functionality compatibility with complex peptides/proteins. In addition, this method can be used for incorporating deuterium into the peptides after cysteine desulfurization by running the reaction in D2O buffer. Moreover, this method enables the clean desulfurization of peptides carrying post‐translational modifications, such as phosphorylation and crotonylation. The effectiveness of this method has been demonstrated by the synthesis of the cyclic peptides dichotomin C and E and synthetic proteins, including ubiquitin, γ‐synuclein, and histone H2A.

P-B Desulfurization: An Enabling Method for Protein Chemical Synthesis and Site-Specific Deuteration.

Cysteine-mediated native chemical ligation is a powerful method for protein chemical synthesis. Herein, we report an unprecedentedly mild system (TCEP/NaBH4 or TCEP/LiBEt3 H; TCEP=tris(2-carboxyethyl)phosphine) for chemoselective peptide desulfurization to achieve effective protein synthesis via the native chemical ligation-desulfurization approach. This method, termed P-B desulfurization, features usage of common reagents, simplicity of operation, robustness, high yields, clean conversion, and versatile functionality compatibility with complex peptides/proteins. In addition, this method can be used for incorporating deuterium into the peptides after cysteine desulfurization by running the reaction in D2 O buffer. Moreover, this method enables the clean desulfurization of peptides carrying post-translational modifications, such as phosphorylation and crotonylation. The effectiveness of this method has been demonstrated by the synthesis of the cyclic peptides dichotomin C and E and synthetic proteins, including ubiquitin, γ-synuclein, and histone H2A.

Determination of Molecular Subtypes of Diffuse Large B-Cell Lymphoma Using a Reverse Transcriptase Multiplex Ligation-Dependent Probe Amplification Classifier: A CALYM Study

Diffuse large B-cell lymphoma (DLBCL) is the most common non-Hodgkin lymphoma. It includes three major subtypes termed germinal center B-cell-like, activated B-cell-like, and primary mediastinal B-cell lymphoma. With the emergence of novel targeted therapies, accurate methods capable of interrogating this cell-of-origin classification should soon become essential in the clinics. To address this issue, we developed a novel gene expression profiling DLBCL classifier based on reverse transcriptase multiplex ligation-dependent probe amplification. This assay simultaneously evaluates the expression of 21 markers, to differentiate primary mediastinal B-cell lymphoma, activated B-cell-like, germinal center B-cell-like, and also Epstein-Barr virus-positive DLBCLs. It was trained using 70 paraffin-embedded biopsies and validated using >160 independent samples. Compared with a reference classification established from Affymetrix U133+2 data, reverse transcriptase multiplex ligation-dependent probe amplification classified 85.0% samples into the expected subtype, comparing favorably with current diagnostic methods. This assay also proved to be highly efficient in detecting the MYD88 L265P mutation, even in archival paraffin-embedded tissues. This reliable, rapid, and cost-effective method uses common instruments and reagents and could thus easily be implemented into routine diagnosis workflows, to improve the management of these aggressive tumors.

Controllable Sulfoxidation and Sulfenylation with Organic Thiosulfate Salts via Dual Electron- and Energy-Transfer Photocatalysis

Sulfoxides and sulfides are two important functional groups in organic molecules, containing different valence states of sulfur. Both sulfoxidation and sulfenylation with common sulfurating reagents were successfully tuned via a facile variation of the atmosphere under photocatalyzed conditions. The sulfoxidation and sulfenylation transformations involved tandem electron-/energy-transfer and single-electron-transfer processes, respectively. Late-stage sulfoxidation for pharmaceuticals and sugar derivatives was established to be highly compatible. Divergent formal syntheses of sulfoxide/sulfide-containing marketed pharmaceuticals were switchably implemented. Gram-scale operations further demonstrated the practicability of the protocol.

Experimental research of the impact of the dosing of chemical reagents on the dynamic behavior of regulation system of cycle chemistry

Organization of reliable chemical control for maintaining cycle chemistry is one of the most important problems to be solved at the present time the design and operation of thermal power plants.To maintain optimal parameters of cycle chemistry are used automated chemical control system and regulation system of dosing chemical reagents. Reliability and stability analyzer readings largely determine the reliability of the water cycle chemistry. Now the most common reagents are ammonia, alkali and film-forming amines.In this paper are presented the results of studies of the impact of concentration and composition of chemical reagents for readings stability of automatic analyzers and transients time of control systems for cycles chemistry.Research of the impact of chemical reagents on the dynamic behavior of regulation system for cycle chemistry was conducted at the experimental facility of the Department of thermal power stations of the Moscow Engineering Institute. This experimental facility is model of the work of regulation system for cycle chemistry close to the actual conditions on the energy facilities CHP.Analysis of results of the impact of chemical reagent on the dynamic behavior of ammonia and film forming amines dosing systems showed that the film-forming amines dosing system is more inertia. This emphasizes the transition process of the system, in which a half times longer dosing of ammonia. Results of the study can be used to improve the monitoring systems of water chemical treatment.

An efficient synthesis of indolo[2,3-b]quinoline guanidine derivatives with their in vitro and in vivo study

An optimization of the guanidylation process by verifying the efficacy of common guanylation reagents in order to obtain the guanidine derivatives of indolo[2,3-b]quinoline has been performed. As a result, a high-yield procedure using N,N′-di-Boc-N′′-triflylguanidine was applied to synthesize the guanidine derivative of indolo[2,3-b]quinoline 1 in a gram scale for specific in vitro and in vivo biological research. Extensive studies on the antiproliferative activity against eight human tumor cell lines were completed. Compound 1 revealed the highest activity against A549 lung adenocarcinoma and MCF7 breast cancer cell lines. Thus, 1 was evaluated for the in vivo anticancer activity against 4T1 mammary gland carcinoma and KLN205 murine lung carcinoma in mouse models. The anticancer effect was observed in the KLN205 model with a 37% tumor growth inhibition at the 20 mg/kg dose. This anticancer activity of 1 was comparable to that of cyclophosphamide which inhibited murine lung tumor growth in the range of 27–43% at the dose of 100 mg/kg. The biochemistry research after 1 admission, including measurements of blood parameters like alanine aminotransferase, aspartate aminotransferase, lactate dehydrogenase, and urea and creatinine, were also performed.

Alternate HPLC method for the analysis of tetrabutylammonium hydroxide in [18F]fluorodeoxythymidine (FLT)

ABSTRACTTetrabutylammonium hydroxide is a common reagent used in the synthesis of [18F]Fluorodeoxythymidine (FLT) for positron emission tomography imaging. The British Pharmacopeia monograph for the analysis of [18F]FLT was released in 2015 incorporating a HPLC method for the analysis of tetrabutylammonium hydroxide. We describe alternate HPLC conditions that mitigate the challenges in fulfilling the system suitability requirements such as signal to noise and symmetry factor that are specified in the monograph. Our method was validated by analyzing tetrabutylammonium hydroxide at a range of concentrations to determine the linearity (R2 = 0.994), accuracy (≤9.2%) and precision (≤3.2%). Our method does not affect the analysis of [18F]FLT and it complies with all requirements in the BP monograph.

Blood or not blood—That is the question. A non-destructive method for the detection of blood-contaminated fingermarks

Working in crime scenes presents a challenge to the forensic scientist, as some surfaces, such as floors and walls, cannot be transferred to the lab for further development and must, therefore, be processed at the crime scene itself. Two main types of latent fingermarks may be encountered in crime scenes: amino acids based and blood contaminated. One of the most common reagents, which are able to develop both types of fingermarks on porous surfaces, is ninhydrin. As blood contaminated fingermarks may be crucial in connecting the suspect to the crime it is important to be able to distinguish between them and natural fingermarks. More than a decade of experience in crime scene investigations led to the understanding that there is a clear visual distinction between natural and blood contaminated fingermarks that are developed by ninhydrin. This study attempted to translate the visual difference into a mobile, non-destructive spectrophotometric method that can be used in crime scenes. Three independent spectrophotometric approaches were examined. The first showed a clear difference between the UV–vis spectra of the solution of blood and ninhydrin versus that of Ruhemann’s purple. The second introduced another method in the solid phase to better simulate a real exhibit found in crime scenes. Once establishing the scientific foundation for the visible difference, a third technique for colour measurements was used in order to provide a potentially fast, quantitative, accurate and non-destructive field test for blood determination at the crime scene.

Development of Time-course Oxygen Binding Analysis for Hemoglobin-based Oxygen Carriers.

Developing blood substitutes is in urgent demand for chronic blood shortage all over the world. In this connection, the oxygen binding behavior of hemoglobin-based oxygen carriers (HBOCs) is one of the most important characteristics. However, present methods available for estimating oxygen binding behavior have need of expensive apparatus, and also are not suitable for high-throughput and the time-course analysis. To overcome these problems, we proposed a simple analysis method for the time-course oxygen binding behavior of HBOCs, which employs a general UV-Vis microplate reader and a common reagent, sodium dithionite, as a reductant for HBOCs and an oxygen scavenger. Our method enabled time-course oxygen binding behavior analysis of HBOCs in a simple manner, and obtained data corresponding with those by the conventional method. Thus, our developed method will accelerate the development of HBOCs due to easy oxygen binding analysis.

Gas-sensing properties and in-situ diffuse-reflectance Fourier-transform infrared spectroscopy study of diethyl ether adsorption and reactions on SnO2 film

Abstract Diethyl ether is a common industrial reagent and medical anesthetic. It is necessary to carry out real-time monitoring of this molecule due to its harmful effects on human health. In this paper, a highly sensitive diethyl ether SnO2 gas-sensing material has been prepared by a sol-gel method. The gas sensitivity was tested by a home-made gas-sensing equipment. The surface adsorption and reaction processes between the SnO2 gas-sensing film and the diethyl ether have been studied by in situ diffuse-reflectance Fourier-transform infrared spectroscopy (DRFT-IR) at different temperatures. The results show that the SnO2 gas-sensing material has high sensitivity to diethyl ether, and the lowest detection limit can reach 1 ppm. Furthermore, ethyl (CH3CH2●), oxoethyl (CH3CH2O●), ethanol (CH3CH2OH), formaldehyde (HCHO), acetaldehyde (CH3CHO), ethylene (C2H4), H2O and CO2 surface species are formed during diethyl ether adsorption at different temperatures. A possible mechanism of the reaction process is discussed.

Sectioning Mammary Gland Whole Mounts for Lesion Identification.

Normal mammary gland development may be altered by exposure to environmental toxicants and pharmaceutical products, excessive exposure to hormones, and genetic alterations. Mammary gland whole mounts are an inexpensive method to capture the progression of morphological changes that may arise after exposure. However, in later life, when abnormalities are more prone to develop, sole reliance on this one method may not always provide enough information to make a proper diagnosis of the abnormality. Historically, in chemical test guideline studies, a single mammary gland is removed at necropsy and prepared as a hematoxylin and eosin (H&E)-stained section. The incorporation of contralateral mammary whole-mount collection and analysis decreases the likelihood of a false-negative assessment. Evaluation of the whole mount is limited by the presence of one or two entire mammary glands on a slide, and in some cases, the abnormalities observed in the whole mount are not uniformly represented in the H&E section. The goal of this study was to develop a protocol for converting coverslipped mammary whole mounts to H&E-stained sections so that lesions that would otherwise have been missed or that are difficult to diagnose can be identified. Here, we detail a method to produce a high-quality, paraffin-embedded H&E section from a mammary gland that was initially prepared as a whole mount. In comparison to a tissue that was intentionally prepared for H&E sectioning, the whole mount requires additional preparation for tissue removal and processing. However, this method is considered inexpensive, as it requires common lab reagents and little additional time. As a result, this method can provide invaluable information on how chemical and environmental exposures alter normal mammary development, as well as display changes that occur because of genetic modifications.