Standard loading controls are not reliable for Western blot quantification across brain development or in pathological conditions

A frequently utilized method of data quantification in Western blot analysis is comparison of the protein of interest with a house keeping gene or control protein. Commonly used proteins include β‐actin, glyceraldehyde 3 phosphate dehydrogenase (GAPDH), and α‐tubulin. Various reliability issues have been raised when using this technique for data analysis—particularly when investigating protein expression changes during development and in disease states. In this study, we have demonstrated that β‐actin, GAPDH, and α‐tubulin are not appropriate controls in the study of development and hypoxic‐ischemic induced damage in the piglet brain. We have also shown that using an in‐house pooled standard, loaded on all blots is a reliable method for controlling interassay variability and data normalization in protein expression analysis.     

β‐Actin as a loading control: Less than 2 μg of total protein should be loaded

For the purpose of data normalization in Western blot analysis, journal editors and reviewers usually require authors to reprobe the Western blot membrane with a β‐actin‐specific antibody after detecting the target protein. In most cases, however, β‐actin is overloaded, which results in a failure to detect differences in protein loading. In this study, we attempted to optimize the amount of protein loaded for β‐actin detection to permit suitable Western blot analysis data normalization. Our data suggest that less than 2 μg of total protein should be loaded when β‐actin is used as a loading control. We also suggest avoiding reprobing the membrane with a β‐actin‐specific antibody.     

Western blot normalization: Time to choose a proper loading control seriously

Recent research has questioned the validity of housekeeping proteins in Western blot. Our present study proposed new ideas for Western blot normalization that improved the reproducibility of scientific research. We used the Gene Expression Omnibus (GEO) database and the web tool GEO2R to exclude unstable housekeeping genes quickly. In ischemic heart tissues, actin and tubulin changed significantly, whereas no statistically significant changes were observed in the expression of genes relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Besides, the reliability of GAPDH was further examined by Western blot. Additionally, unstable housekeeping genes were found in other animal models of cardiovascular medicine. We also found that sodium dodecyl sulfate and temperature significantly impacted the results of Ponceau S staining. Membranes stained with Ponceau S after immunodetection could avoid this interference, and the coefficients of variation for post-immunodetection staining are lower than those produced by GAPDH immunodetection. Overall, we described a new use of differential gene expression analysis and proposed a modified Ponceau S staining method, which provided researchers with a proper loading control for Western blot and hence could improve reproducibility in research.     

Beta-actin is not a reliable loading control in Western blot analysis

Beta-actin is often used as a loading control in Western blot analysis. We analyzed the ability of beta-actin-specific antibodies to recognize differences in protein loading. We found that, at higher total protein loads as required for the detection of low-abundance proteins, beta-actin-specific antibodies failed to distinguish differences in actin protein levels. Diluting the antibody working solution or changing the incubation time had little effect on this phenomenon. This shows that beta-Actin is not a reliable loading control in Western blot analysis. In general, it appeared that, at longer incubation times, antibodies seem to be less able to pick up differences in the level of its target protein.     

Β‐actin does not show the characteristics of a reference protein in human cortex

Levels of a reference protein must be the same as a proportion of total protein in all tissues and, in the study of human diseases, cannot vary with factors such as age, gender or disease pathophysiology. It is increasingly apparent that there may be few, if any, proteins that display the characteristics of a reference protein within the human central nervous system (CNS). To begin to challenge this hypothesis, we used Western blotting to compare variance in levels of the “gold standard” reference protein, β‐actin, in Brodmann's area 9 from 194 subjects to variance of total transferred protein measured as intensity of Ponceau S staining. The coefficient of variance of sum intensity measurements for β‐actin levels across all donors was 47% compared to 24 and 27% for the sum intensity of Ponceau S staining measured using two different detection techniques. These data strongly suggest that the level of β‐actin, proportional to total protein, is not constant in human cortex which raises further doubt about the use of reference proteins to normalise data in human CNS studies. Considering our data, we suggest an alternative approach to presenting data from Western blotting of human CNS.     

Direct Evidence for a Peroxide Intermediate and a Reactive Enzyme–Substrate–Dioxygen Configuration in a Cofactor‐free Oxidase

Cofactor‐free oxidases and oxygenases promote and control the reactivity of O₂ with limited chemical tools at their disposal. Their mechanism of action is not completely understood and structural information is not available for any of the reaction intermediates. Near‐atomic resolution crystallography supported by in crystallo Raman spectroscopy and QM/MM calculations showed unambiguously that the archetypical cofactor‐free uricase catalyzes uric acid degradation via a C5(S)‐(hydro)peroxide intermediate. Low X‐ray doses break specifically the intermediate C5? OO(H) bond at 100 K, thus releasing O₂ in situ, which is trapped above the substrate radical. The dose‐dependent rate of bond rupture followed by combined crystallographic and Raman analysis indicates that ionizing radiation kick‐starts both peroxide decomposition and its regeneration. Peroxidation can be explained by a mechanism in which the substrate radical recombines with superoxide transiently produced in the active site.     

L‐Proline‐based Phosphamides as a New Kind of Organocatalyst for Asymmetric Direct Aldol Reactions

Four L‐proline‐based phosphamides were designed and synthesized as a new kind of organocatalyst. Their catalytic activities for asymmetric direct aldol reactions were evaluated. Among them, 3a with 10 mol% catalyst loading afforded moderate to good yields and up to 99% ee.     

Development of a method using high‐performance liquid chromatographic fingerprint and multi‐ingredients quantitative analysis for the quality control of Yangxinshi Pian

A simple and reliable method of high‐performance liquid chromatography with diode array detection method was developed for fingerprint analysis and simultaneous determination of six compounds including puerarin, salvianolic acid B, berberine hydrochloride, palmatine chloride, dehydrocorydaline, and icariin in the Chinese medicine preparation Yangxinshi Pian. The separation was performed on an Agilent Eclipse XDB‐C₁₈ reserved‐phase column (250 mm × 4.6mm I.D., 5 μm) using gradient elution with 50 mmol/L monopotassium phosphate aqueous solution and methanol as mobile phase at a flow rate of 1.0 mL/min. The column operating temperature was set at 30°C, and the detection wavelength was 280 nm. The method was validated by linearity, precision, accuracy, stability, and recovery. For fingerprint analysis, 25 peaks were selected as the common peaks, and four kinds of similarities including cosine similarity (S), ratio of similarity (S′), projection content similarity (C), and content similarity (P) were applied to evaluate the quality consistency of different batches of Yangxinshi Pian. The results showed that the developed method was an efficient tool for quality evaluation of Yangxinshi Pian.     

Intrinsic Electrocatalysis of RNA as a Label‐free and Reagent‐less Tool for Detection of MicroRNAs

We show for the first time that RNA catalyzes hydrogen evolution reaction at mercury‐containing electrodes. We previously showed that DNA is electrocatalytically active, and so we compared heights and potentials of the RNA chronopotentimetric stripping (CPS) peaks with analogous signals of DNA of the same sequence and found out they were very similar. RNA peaks showed differences depending on the RNA base composition. Catalytic nature of CPS peak enabled detection of 25 pM microRNA in electrochemical cell, or 500 pM microRNA in a 5 μL solution drop (corresponding to 2.5 fmole of microRNA). This finding opens the door for simple, label‐free and reagent‐less analysis of low concentrations of RNA molecules.     

Development of a sensitive GC‐C‐IRMS method for the analysis of androgens

The administration of anabolic steroids is one of the most important issues in doping control and is detectable through a change in the carbon isotopic composition of testosterone and/or its metabolites. Gas chromatography‐combustion‐isotope ratio mass spectrometry (GC‐C‐IRMS), however, remains a very laborious and expensive technique and substantial amounts of urine are needed to meet the sensitivity requirements of the IRMS. This can be problematic because only a limited amount of urine is available for anti‐doping analysis on a broad spectrum of substances. In this work we introduce a new type of injection that increases the sensitivity of GC‐C‐IRMS by a factor of 13 and reduces the limit of detection, simply by using solvent vent injections instead of splitless injection. This drastically reduces the amount of urine required. On top of that, by only changing the injection technique, the detection parameters of the IRMS are not affected and there is no loss in linearity. Copyright © 2012 John Wiley & Sons, Ltd.     

Electrodeposition CaCO3 Nanoparticles‐chitosan Composite Film on Carbon Ionic Liquid Electrode as a Platform for Hemoglobin Electrochemical Biosensor

By one‐step co‐electrodeposition CaCO₃ nanoparticles‐chitosan composite film on carbon ionic liquid electrode (CILE), and then by spreading the composition of hemoglobin (Hb) and chitosan on the nanoCaCO₃‐chi/CILE, a Hb‐chi/nanoCaCO₃‐chi/CILE was fabricated and the direct electrochemistry and electrocatalysis of Hb at the electrode was investigated. The electrochemical impedance spectroscopy of the modified electrode showed the electron transfer resistance was 1166 Ω. Investigation results of cyclic voltammetrys showed a pair of well‐defined and quasireversible redox peak of Hb with the formal potentials of ‐0.295 V (vs. SCE) in 0.1 mol·L^‐1 pH 7.0 PBS; the response time of the reduction peak currents of Hb was lower than 3s; a linear range for determination of H₂O₂ was from 5.0 μmol·L^‐1 to 1.3 mmol·L^‐1 with a detection limit of 1.6 μmol·L^‐1 (S/N = 3) and a sensitivity of 0.16 A·M^‐1·cm^‐2; the electron transfer rate constant and the apparent Michaelis‐Menten constant of Hb were 1.98 s^‐1 and 0.81 mmol·L^‐1, respectively. As a result, the case of the one‐step co‐electrodeposition and the promising feature of biocomposite could serve as a versatile platform for the fabrication of electrochemical biosensors.     

Alumina as a Support and Catalyst for the Synthesis of Benzothiazoles in Solvent‐free Condition

Arylbenzothiazole ( 4 ) and benzothiazolylcoumarins ( 5 ) were prepared by the condensation of 2‐aminothiophenol ( 1 ) and hydroxybenzaldehydes ( 2 ) and 3‐ethoxycarbonylcoumarinnin ( 3 ), respectively, in solvent‐free condition. Of the solid support used, γ‐Al₂O₃ demonstrated the best activity. A linear regression line obtained from correlation between relative formation rate and Hammett constants with slope of 0.653 suggested that this condensation process is less sensitive to the substituents on the phenyl ring due to low efficacy of polarization in the solid state. In general, good yields (>80% yield) were obtained for the formation of 4 . Conversely, yields of 5 were poor, because of γ‐Al₂O₃ catalytic decomposition of 3 to form 2 (without P₂O₅) and coumarin (with P₂O₅).     

Application of Si‐doped graphene as a metal‐free catalyst for decomposition of formic acid: A theoretical study

Using density functional theory calculations, the adsorption and catalytic decomposition of formic acid (HCOOH) over Si‐doped graphene are investigated. For the stable adsorption geometries of HCOOH over Si‐doped graphene, the electronic structure properties are analyzed by adsorption energy, density of states, and charge density difference. A comparison of the reaction pathways reveals that both dehydration and dehydrogenation of HCOOH can occur over Si‐doped graphene. The estimated reaction energies and the activation barriers suggest that for the dehydration of HCOOH on the Si‐doped graphene, the rate‐controlling step is H + OH → H₂O reaction. For the dehydrogenation of HCOOH, the rate‐determining step is the breaking of the C? H bond of the HCOO group to form the CO₂ molecule and the atomic H. Our results reveal that the low cost Si‐doped graphene can be used as an efficient nonmetal catalyst for O? H bond cleavage of HCOOH. © 2015 Wiley Periodicals, Inc.     

Sulfamic Acid‐functionalized Nano‐titanium dioxide as a Novel and Highly Efficient Heterogeneous Nanocatalyst for One‐pot and Solvent‐free Synthesis of Hexahydroquinolines

Hexahydroquinolines are synthesized via one‐pot, four‐component condensation of 1,3‐cyclic diketones, aromatic aldehydes, malononitrile, and ammonium acetate in the presence of sulfamic acid‐functionalized nano‐titanium dioxide as a novel type of heterogeneous nanocatalyst. The optimized reaction condition is achieved using response surface method (Box–Behnken design [BBD ]). Operational simplicity, low cost, nontoxic nature, and high reusability of the catalyst together with simple work‐up and excellent product yields are the main advantages of this methodology. Also, the nanocatalyst can be recovered easily by centrifugation and reused efficiently for at least seven consecutive runs.     

Facilitating quality control for spectra assignments of small organic molecules: nmrshiftdb2 – a free in‐house NMR database with integrated LIMS for academic service laboratories

nmrshiftdb2 supports with its laboratory information management system the integration of an electronic lab administration and management into academic NMR facilities. Also, it offers the setup of a local database, while full access to nmrshiftdb2's World Wide Web database is granted. This freely available system allows on the one hand the submission of orders for measurement, transfers recorded data automatically or manually, and enables download of spectra via web interface, as well as the integrated access to prediction, search, and assignment tools of the NMR database for lab users. On the other hand, for the staff and lab administration, flow of all orders can be supervised; administrative tools also include user and hardware management, a statistic functionality for accounting purposes, and a ‘QuickCheck’ function for assignment control, to facilitate quality control of assignments submitted to the (local) database. Laboratory information management system and database are based on a web interface as front end and are therefore independent of the operating system in use. Copyright © 2015 John Wiley & Sons, Ltd.     

Carbon Dots as a Promising Green Photocatalyst for Free Radical and ATRP‐Based Radical Photopolymerization with Blue LEDs

Carbon dots (CDs) have been used for the first time as a sensitizer to initiate and activate free radical and controlled radical polymerization, respectively, based on an ATRP protocol with blue LEDs. Consideration of diverse heteroatom‐doped CDs indicated that N‐doped CDs could serve as an effective photocatalyst and photosensitizer in combination with LEDs emitting either at 405 nm or 470 nm. Free radical polymerization was initiated by combining the CDs with an iodonium or sulfonium salt in tri(propylene glycol) diacrylate. Polymerization of methyl methacrylate (MMA) by photo‐induced ATRP was achieved with CDs and ethyl α‐bromophenylacetate using Cu^II as catalyst in the ppm range. The polymers obtained showed temporal control, narrower dispersity ?1.5, and chain‐end fidelity. The first‐order kinetics and ON/OFF experiments additionally gave evidence of the constant concentration of polymer radicals. No remarkable cytotoxic activity was observed for the CDs, underlining their biocompatibility.     

Eosin Y as a Direct Hydrogen‐Atom Transfer Photocatalyst for the Functionalization of C−H Bonds

Eosin Y, a well‐known economical alternative to metal catalysts in visible‐light‐driven single‐electron transfer‐based organic transformations, can behave as an effective direct hydrogen‐atom transfer catalyst for C?H activation. Using the alkylation of C?H bonds with electron‐deficient alkenes as a model study revealed an extremely broad substrate scope, enabling easy access to a variety of important synthons. This eosin Y‐based photocatalytic hydrogen‐atom transfer strategy is promising for diverse functionalization of a wide range of native C?H bonds in a green and sustainable manner.     

Qualitative and quantitative analysis of multiple components for quality control of Deng‐Zhan‐Sheng‐Mai capsules by ultra high performance liquid chromatography tandem mass spectrometry method coupled with chemometrics

Deng‐Zhan‐Sheng‐Mai capsules are a well‐known traditional Chinese patent medicine that was developed in China for the treatment of ischemic stroke. Its quality control focuses on Erigerontis Herba but ignores the contributions of Ginseng Radix et Rhizoma, Schisandrae Chinensis Fructus, and Ophiopogonis Radix. To improve the quality standards for this medicine, this work reports the application of a systematic ultra high performance liquid chromatography tandem mass spectrometric method coupled with chemometrics. Three qualitative and quantitative parameters are established for the evaluation of quality: chemical profiling, the relationship between the contents of 18 compounds and the antioxidant activity, and chemometric analysis. A total of 55 compounds, including 20 phenolic acids, 10 flavonoids, 15 saponins, and 10 lignans, were identified. The method for the quantitative determination of the aforementioned 18 compounds was validated. The limit of quantification ranged from 0.13 to 9.60 ng/mL. The overall recoveries ranged from 95.31 to 103.54%. Hierarchical cluster analysis and principal component analysis were applied to the data of 18 components in ten batches of samples. Nine compounds, including scutellarin, 3,5‐O‐dicaffeoylquinic acid, 4,5‐O‐dicaffeoylquinic acid, ginsenoside Rb1, ginsenoside Re, ginsenoside Rg1, ophiopogonin D, schisandrin, and schisandrol B, are suggested as chemical markers for evaluating the quality.     

Cobalt nanoparticles anchored to porous silicon as a novel modifier for the construction of enzyme‐free hydrogen peroxide screen‐printed sensor

In this work, a screen‐printed carbon electrode (SPCE) was modified with a cobalt/porous silicon (Co@PSi) nanocomposite powder to develop a nonenzymatic sensor for the detection of hydrogen peroxide. The Co@PSi nanocomposite was synthesized through the chemical reaction between silicon powder in a HF/HNO₃ solution and cobalt cations. In this process, cobalt nanoparticles were anchored on the porous silicon. The structure and morphology of the synthesized nanocomposite were investigated by X‐ray diffraction, Fourier transform infrared spectroscopy, X‐ray photoemission spectroscopy, energy dispersive X‐ray spectroscopy, and field‐emission scanning electron microscopy. The constructed nonenzymatic, screen‐printed sensors based on the Co@PSi nanocomposite showed perfect electrocatalytic oxidation response to hydrogen peroxide over the range 1–170 and 170–3,770 μmol/L with the limit of detection of 0.8 μmol/L. In addition, the Co@PSi‐SPCE sensor exhibited good selectivity for the determination of H₂O₂ in the presence of common interfering species including glucose, ascorbic acid, uric acid, dopamine, nitrate, and nitrite ions. The constructed electrochemical sensor was successfully used for the determination of H₂O₂ in real samples.