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Analytical Biochemistry

Alexander Kyrychenko, Mykola V Rodnin, Chiranjib Ghatak, Alexey S Ladokhin
The variability of the orientation factor is a long-standing challenge in converting FRET efficiency measurements into donor-acceptor distances. We propose the use of molecular dynamics (MD) simulations to characterize orientation distributions and thus improve the accuracy of distance measurements. Here, we test this approach by comparing experimental and simulated FRET efficiencies for a model donor-acceptor pair of enhanced cyan and enhanced yellow FPs connected by a flexible linker. Several spectroscopic techniques were used to characterize FRET in solution...
January 17, 2017: Analytical Biochemistry
Kwabena Sarpong, Ron Bose
A major challenge in attaching fluorophores or other handles to proteins is the availability of a site-specific labeling strategy that provides stoichiometric modification without compromising protein integrity. We developed a simple approach that combines TEV protease cleavage, sortase modification and affinity purification to N-terminally label proteins. To achieve stoichiometrically-labeled protein, we included a short affinity tag in the fluorophore-containing peptide for post-labeling purification of the modified protein...
January 11, 2017: Analytical Biochemistry
Mirela Sarbu, Liana Dehelean, Cristian V A Munteanu, Željka Vukelić, Alina D Zamfir
The gangliosides (GGs) of the central nervous system (CNS) exhibit age and topographic specificity and these patterns may correlate with the functions and pathologies of the brain regions. Here, chloroform extraction, nanoelectrospray (nanoESI) negative ionization, together with Orbitrap high resolution mass spectrometry (MS) determined the topographic and age-related GG specificity in normal adult human brain. Mapping of GG mixtures extracted from 20 to 82 year old frontal and occipital lobes revealed besides a decrease in the GG number with age, a variability of sialylation degree within the brain regions...
January 11, 2017: Analytical Biochemistry
William J Griffiths, Jonas Abdel-Khalik, Eylan Yutuc, Alwena H Morgan, Ian Gilmore, Thomas Hearn, Yuqin Wang
Cholesterolomics can be regarded as the identification and quantification of cholesterol, its precursors post squalene, and metabolites of cholesterol and of its precursors, in a biological sample. These molecules include 1,25-dihydroxyvitamin D3, steroid hormones and bile acids and intermediates in their respective biosynthetic pathways. In this short article we will concentrate our attention on intermediates in bile acid biosynthesis pathways, in particular oxysterols and cholestenoic acids. These molecular classes are implicated in the aetiology of a diverse array of diseases including autoimmune disease, Parkinson's disease, motor neuron disease, breast cancer, the lysosomal storage disease Niemann-Pick type C and the autosomal recessive disorder Smith-Lemli-Opitz syndrome...
January 10, 2017: Analytical Biochemistry
Firas S Midani, Michelle L Wynn, Santiago Schnell
The use of isotopically labeled tracer substrates is an experimental approach for measuring in vivo and in vitro intracellular metabolic dynamics. Stable isotopes that alter the mass but not the chemical behavior of a molecule are commonly used in isotope tracer studies. Because stable isotopes of some atoms naturally occur at non-negligible abundances, it is important to account for the natural abundance of these isotopes when analyzing data from isotope labeling experiments. Specifically, a distinction must be made between isotopes introduced experimentally via an isotopically labeled tracer and the isotopes naturally present at the start of an experiment...
January 10, 2017: Analytical Biochemistry
Svetlana V Kalinichenko, Mikhail V Shepelev, Igor V Korobko
Agarose gel electrophoresis with subsequent DNA extraction from gel is routinely used for DNA fragment isolation after plasmid DNA digestion. We describe a gel-less method for DNA fragment isolation after plasmid DNA digestion which is based on in-solution negative selection through depletion of vector backbone bearing LoxP sites by sorption on solid phase-immobilized mutated Cre recombinase. The method might be especially useful in preparation of DNA fragments for transgenic animal generation where residual agarose presence is a concern, and DNA fragments are frequently large in size and thus might be mechanically damaged during purification with conventional affinity-based gel extraction methods...
January 9, 2017: Analytical Biochemistry
Phil Lee, Peter Adany, In-Young Choi
Accurate quantitative metabolic imaging of the brain presents significant challenges due to the complexity and heterogeneity of its structures and compositions with distinct compartmentations of brain tissue types (e.g., gray and white matter). The brain is compartmentalized into various regions based on their unique functions and locations. In vivo magnetic resonance spectroscopy (MRS) techniques allow non-invasive measurements of neurochemicals in either single voxel or multiple voxels, yet the spatial resolution and detection sensitivity of MRS are significantly lower compared with MRI...
January 9, 2017: Analytical Biochemistry
Sara Samuei, Jila Fakkar, Zolfaghar Rezvani, Ashkan Shomali, Biuck Habibi
In the present work, a novel nanocomposite based on the graphene quantum dots and CoNiAl-layered double-hydroxide was successfully synthesized by co-precipitation method. To achieve the morphological, structural and compositional information, the resulted nanocomposite was characterized by scanning electron microscopy X-ray diffraction, thermal gravimetric analysis, Fourier transform infrared spectroscopy, and photoluminescence. Then, the nanocomposite was used as a modifier to fabricate a modified carbon paste electrode as a non-enzymatic sensor for glucose determination...
January 9, 2017: Analytical Biochemistry
Julia Raykin, Eric Snider, Sruti Bheri, John Mulvihill, C Ross Ethier
Gelatinase zymography is a commonly used laboratory procedure; however, variability in sample loading and concentration reduce the accuracy of quantitative results obtained from this technique. To facilitate normalization of gelatinase activity by loaded protein amount, we developed a protocol using the trihalocompound 2,2,2-trichloroethanol to allow for gelatin zymography and total protein labeling within the same gel. We showed that detected protein levels increased linearly with loading, and describe a loading concentration range over which normalized gelatinase activity was constant...
January 6, 2017: Analytical Biochemistry
Péter Polyák, Piroska Rácz, Péter Rózsa, Gergely N Nagy, Beáta G Vértessy, Béla Pukánszky
A novel method was introduced for the quantitative determination of substances in aqueous solutions by using the evaporative light scattering (ELS) detector of a high performance liquid chromatograph (HPLC). The principle of the measurement is the different equilibrium vapor pressure of the solvent and the analyte resulting in decreasing evaporation rate, larger droplets and stronger signal with increasing concentration. The new technique based on vapor pressure analysis was validated with traditional UV-Vis detection carried out with a diode array detector (DAD)...
January 6, 2017: Analytical Biochemistry
Fantao Meng, Abdu I Alayash
The role of hemoglobin (Hb) redox forms in tissue and organ toxicities remain ambiguous despite the well-documented contribution of Hb redox reactivity to cellular and subcellular oxidative changes. Moreover, several recent studies, in which Hb toxicity were investigated, have shown conflicting outcomes. Uncertainties over the potential role of these species may in part be due to the protein preparation method of choice, the use of published extinction coefficients and the lack of suitable controls for Hb oxidation and heme loss...
January 6, 2017: Analytical Biochemistry
Xin Zhang, Sergey Voronov, Nesredin Mussa, Zhengjian Li
Imaged Capillary Isoelectric Focusing (icIEF) has been used as primary method for charge variants analysis of therapeutic antibodies and proteins [1], [9]. Proteins tend to precipitate around their pI values during focusing [14], which directly affects the reproducibility of their charge profiles. Protein concentration, focusing time and various supplementing additives are key parameters to minimize the protein precipitation and aggregation. Urea and sucrose are common additives to reduce protein aggregation, solubilize proteins in sample matrix and therefore improve assay repeatability [15]...
January 6, 2017: Analytical Biochemistry
Zhen Luo, Qin Zhao, Jixiang Liu, Jinfang Liao, Ruogu Peng, Yunting Xi, Zhenjun Diwu
Peroxynitrite (ONOO(-)), a strong oxidant species, is produced by the reaction of nitric oxide (NO) and superoxide (O2(.-)) radicals. It plays an important role as a biological regulator in numbers of physiological and pathological processes. In this study, we developed fluorescence-based real-time quantitative measurements to detect intracellular ONOO(-). The probe DAX-J2 PON Green showed high selectivity toward ONOO(-) over other competing species, and has been successfully applied in microplate reader and flow cytometer to quantitatively measure endogenous ONOO(-) production...
January 4, 2017: Analytical Biochemistry
Ning Zhao, John Spencer, Margaret A Schmitt, John D Fisk
Tuberculosis is the leading cause of death from infectious disease worldwide. The low sensitivity, extended processing time, and high expense of current diagnostics are major challenges to the detection and treatment of tuberculosis. Mycobacterium tuberculosis ornithine transcarbamylase (Mtb OTC, Rv1656) has been identified in the urine of patients with active TB infection and is a promising target for point-of-care diagnostics. Specific binding proteins with low nanomolar affinities for Mtb OTC were selected from a phage display library built upon a hyperthermostable Sso7d scaffold...
January 3, 2017: Analytical Biochemistry
Tomoko Yoshino, Tsuyoshi Tanaka, Seita Nakamura, Ryo Negishi, Nozomi Shionoiri, Masahito Hosokawa, Tadashi Matsunaga
A cell entrapment device consisting of a microcavity array was used to analyze the deformability of MCF-10 human breast epithelial and MCF-7 human breast cancer cell lines by confocal laser scanning microscopy. Entrapment of up to 8 × 10(3) cells was achieved within 3 min. Protrusions were formed at the bottom surface of the array with a pore size of 3 μm. Protrusion length increased at higher filtration pressures and could be used to distinguish between MCF-7 and MCF-10 cells. These results indicate that our system is useful for high-throughput deformability analysis of cancer cells, which can provide insight into the mechanisms underlying tumor cell malignancy...
January 3, 2017: Analytical Biochemistry
Kazutaka Araki, Ryo Ushioda, Hidewo Kusano, Riko Tanaka, Tomohisa Hatta, Kazuhiko Fukui, Kazuhiro Nagata, Tohru Natsume
Thiol-based redox control is among the most important mechanisms for maintaining cellular redox homeostasis, with essential participation of cysteine thiols of oxidoreductases. To explore cellular redox regulatory networks, direct interactions among active cysteine thiols of oxidoreductases and their targets must be clarified. We applied a recently described thiol-ene crosslinking-based strategy, named divinyl sulfone (DVSF) method, enabling identification of new potential redox relay partners of the cytosolic oxidoreductases thioredoxin (TXN) and thioredoxin domain containing 17 (TXNDC17)...
December 31, 2016: Analytical Biochemistry
Margaret M Elmer-Dixon, Bruce E Bowler
A novel approach to quantification of cardiolipin and DOPC lipid and vesicle concentration that is rapid and inexpensive is described. Traditional approaches to quantifying vesicle concentration destroy sample and are often time consuming. Using common laboratory equipment and software, lipid vesicles were reliably quantified allowing for immediate use without significant sample loss. Once calibrated, only absorbance measurements with a UV-Vis spectrophotometer are necessary as input into a Matlab program, which calculates the corresponding vesicle and lipid concentration...
December 27, 2016: Analytical Biochemistry
Caroline D Rae, Stephen R Williams
We review the transport, synthesis and catabolism of glutathione in the brain as well as its compartmentation and biochemistry in different brain cells. The major reactions involving glutathione are reviewed and the factors limiting its availability in brain cells are discussed. We also describe and critique current methods for measuring glutathione in the human brain using magnetic resonance spectroscopy, and review the literature on glutathione measurements in healthy brains and in neurological, psychiatric, neurodegenerative and neurodevelopmental conditions In summary: Healthy human brain glutathione concentration is ∼1-2 mM, but it varies by brain region, with evidence of gender differences and age effects; in neurological disease glutathione appears reduced in multiple sclerosis, motor neurone disease and epilepsy, while being increased in meningiomas; in psychiatric disease the picture is complex and confounded by methodological differences, regional effects, length of disease and drug-treatment...
December 26, 2016: Analytical Biochemistry
Wolfgang Bogner, Gilbert Hangel, Morteza Esmaeili, Ovidiu C Andronesi
This article reviews the methodological aspects of detecting low-abundant J-coupled metabolites via 1D spectral editing techniques and 2D nuclear magnetic resonance (NMR) methods applied in vivo, in humans, with a focus on the brain. A brief explanation of the basics of J-evolution will be followed by an introduction to 1D spectral editing techniques (e.g., J-difference editing, multiple quantum coherence filtering) and 2D-NMR methods (e.g., correlation spectroscopy, J-resolved spectroscopy). Established and recently developed methods will be discussed and the most commonly edited J-coupled metabolites (e...
December 26, 2016: Analytical Biochemistry
Ana Francisca Soares, Rolf Gruetter, Hongxia Lei
In the brain, glycogen is a source of glucose not only in emergency situations but also during normal brain activity. Altered brain glycogen metabolism is associated with energetic dysregulation in pathological conditions, such as diabetes or epilepsy. Both in humans and animals, brain glycogen levels have been assessed non-invasively by Carbon-13 Magnetic Resonance Spectroscopy ((13)C-MRS) in vivo. With this approach, glycogen synthesis and degradation may be followed in real time, thereby providing valuable insights into brain glycogen dynamics...
December 26, 2016: Analytical Biochemistry
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