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Analytical Biochemistry

V Vasilca, A Sadeghpour, S Rawson, L E Hawke, S A Baldwin, T Wilkinson, D Bannister, V L G Postis, M Rappolt, S P Muench, L J C Jeuken
Screening assays performed against membrane protein targets (e.g. phage display) are hampered by issues arising from protein expression and purification, protein stability in detergent solutions and epitope concealment by detergent micelles. Here, we have studied a fast and simple method to improve screening against membrane proteins: spherical-supported bilayer lipid membranes ("SSBLM"). SSBLMs can be quickly isolated via low-speed centrifugation and redispersed in liquid solutions while presenting the target protein in a native-like lipid environment...
March 12, 2018: Analytical Biochemistry
Yan Lu, Lianjie Meng, Yan Gao, Dongli Liao, Yongxin Li, Yongxing Ai, Yuqin Ma, Cong Yu
Metal ions sensing play critical roles in environmental monitoring and in biology. In this assay, we report the development of a facile fluorometric method for the sensing of Ag+ ions via the in situ formation of metal coordination polymer, based on the selective interactions of GSH with Ag+ . The formation of coordination polymer with net multiple negative charges in an aqueous buffer solution (Tris-HAc, pH 9.0) resulted in aggregation and fluorescence quenching of a cationic perylene probe. The difference in emission intensity spurred us to develop a new strategy for sensing Ag+ ions...
March 12, 2018: Analytical Biochemistry
Yu Lu, Chengjian Wang, Rendan Liu, Wanjun Jin, Yanan Wen, Linjuan Huang, Zhongfu Wang
Sensitive analysis of glycans by liquid chromatography/mass spectrometry is significantly hampered by the lack of chromogenic or fluorescent groups on the glycan structures, as well as, their poor ionization properties. In the present, a heterobifunctional chromogenic reagent 3-amino-1-phenyl-2-pyrazoline-5-ketone (PAP) bearing amino and active methylene groups, which readily reacts with reducing glycans, was used for detection of the pre-column-labeled glycans via high-performance liquid chromatography/electrospray ionization mass spectrometry (HPLC/ESI-MS)...
March 7, 2018: Analytical Biochemistry
Jinan Tuma Sabah, Razauden Mohamed Zulkifli, Shafinaz Shahir, Farediah Ahmed, Mohammed Rafiq Abdul Kadir, Zarita Zakaria
Distinctive bioactivities possessed by luteolin (3', 4', 5, 7-tetrahydroxy-flavone) are advantageous for sundry practical applications. This paper reports the in vitro selection and characterization of single stranded-DNA (ssDNA) aptamers, specific for luteolin (LUT). 76-mer library containing 1015 randomized ssDNA were screened via systematic evolution of ligands by exponential enrichment (SELEX). The recovered ssDNA pool from the 8th round was amplified with unlabeled primers and cloned into PSTBlue-1 vector prior to sequencing...
March 6, 2018: Analytical Biochemistry
G Hawa, Linda Sonnleitner, A Missbichler, A Prinz, G Bauer, C Mauracher
Although Enzyme Linked Immuno Sorbent Assay (ELISA) technology is approaching it's 45th year of existence since first described in 1971, it is still the main diagnostic tool in clinical research and routine diagnostics. However, despite its broad usage it suffers from some drawbacks, limiting its use especially in more advanced assay formats like multiplexing platforms, point of care devices or protein arrays. Those limitations result from the need for an enzyme label, a soluble enzyme substrate, washing steps (multiplexing, point care, arrays) and in some cases also insufficient sensitivity, because the majority of circulating proteins and thus potential biomarkers may be found in lowor sub-picomolar concentrations...
March 5, 2018: Analytical Biochemistry
Chao-Pin Hsiao, Charles Hoppel
Mitochondrial oxidative phosphorylation (OXPHOS) is responsible for producing most of the adenosine triphosphate required by eukaryotic cells. Lymphocytes make up the majority of the peripheral blood mononuclear cells. Peripheral blood mononuclear cells are readily obtainable, providing an ideal sample to monitor systemic changes and understand molecular signaling mechanisms in disease processes. Mitochondrial energy metabolism of lymphocyte has been used to screen for OXPHOS disorders. While there are increasing studies of lymphocyte OXPHOS, few studies examined activity of electron transport chain of lymphocyte mitochondria...
March 2, 2018: Analytical Biochemistry
Harumi Osago, Mikiko Kobayashi-Miura, Yoshifumi Hamasaki, Nobumasa Hara, Mineyoshi Hiyoshi, Mikako Tsuchiya
Articular cartilage comprises collagens, proteoglycans, and glycosaminoglycans (GAGs) together with water, in hyaline matrixes. Articular cartilage is resistant to proteolytic solubilization for comprehensive GAG analyses partly because of assemblies of collagen fibers with thermolabile hydrogen bonds. In this study, we used the heat-stable protease thermolysin to digest collagen in solid articular cartilage at 70 °C and compared the efficiencies of collagen digestion and GAG extraction to those with collagenase digestion at 50 °C...
March 2, 2018: Analytical Biochemistry
Megan C Harwig, Matheus P Viana, John M Egner, Jason J Harwig, Michael E Widlansky, Susanne M Rafelski, R Blake Hill
Mitochondria are found in a variety of shapes, from small round punctate structures to a highly interconnected web. This morphological diversity is important for function, but complicates quantification. Consequently, early quantification efforts relied on various qualitative descriptors that understandably reduce the complexity of the network leading to challenges in consistency across the field. Recent application of state-of-the-art computational tools have resulted in more quantitative approaches. This prospective highlights the implementation of MitoGraph, an open-source image analysis platform for measuring mitochondrial morphology initially optimized for use with Saccharomyces cerevisiae...
March 2, 2018: Analytical Biochemistry
Iva Turyan, Ruth Frenkel, Zoran Sosic
Protein tyrosine sulfation (Tyr-O-SO3) is a common post-translational modification (PTM), which is important for protein function. Absolute quantitation of Tyr-O-SO3 in recombinant therapeutic proteins has been challenging. We report here an MRM method used for absolute quantitation of Tyr-O-SO3 in the hydrolysate of a recombinant Fc-fusion protein. Quantitation is achieved by monitoring the sum of two transitions: the loss of carboxylic acid from tyrosine sulfate (major transition) and sulfate group from tyrosine sulfate sodium salt...
March 2, 2018: Analytical Biochemistry
Huw Barton, Waldir M Berbel-Filho, Sofia Consuegra, Lewis Francis, Chedly Tizaoui, R Steven Conlan, Sofia Rodrigues Teixeira
There is a growing interest in the possible environmental health impact posed by endocrine-disrupting chemicals (EDCs). A challenge to the field of endocrine disruption is that these substances are diverse and may not appear to share any structural similarity other than usually being low molecular mass (<1000 Daltons) compounds. Here we demonstrate the effectiveness of sensor device for the detection of low molecular weight, poorly water soluble, estrogenic compounds E1, E2 and EE2, fabricated by electropolymerization over graphene screen printed electrode (SPE)...
March 1, 2018: Analytical Biochemistry
Mohamad Zandieh, Seyyed Nezam Hosseini, Manouchehr Vossoughi, Maryam Khatami, Sara Abbasian, Ahmad Moshaii
This paper describes the construction of a silver-based LSPR biosensor for endotoxin detection. We used GLAD method to procure reproducible silver nanocolumns. In this work, the silver nanostructures were considerably stabilized by a SAM of MPA, and the limit of detection of biosensor was measured to be 340 pg/ml for endotoxin E. coli. Considering endotoxin B. abortus as the second type of endotoxin contamination in our target samples (HBs-ag produced in Institute Pasteur, Iran), we investigated selectivity of the biosensor in various experiments...
February 28, 2018: Analytical Biochemistry
Cuixia Guo, Yiling Hu, Chunyu Yang, Ankanahalli N Nanjaraj Urs, Yan Zhang
The Fe(II)/2-oxoglutarate-dependent dioxygenases (2-OGDs) catalyze the oxidation of substrates ranging from small molecules to large biomolecules with concomitant oxidation of co-substrate (2-oxoglutarate) into succinate. In the present study, we reported a coupled colorimetric assay that can be generally applied to measure the activities of all members of 2-OGDs family. Succinyl-CoA synthetase is employed as the coupling enzyme to transform succinate produced from 2-OGDs catalysis to form succinyl-CoA with concomitant hydrolysis of ATP to form ADP and orthophosphate...
February 27, 2018: Analytical Biochemistry
Hamid Sedighian, Raheleh Halabian, Jafar Amani, Mohammad Heiat, Ramezan Ali Taheri, Abbas Ali Imani Fooladi
Staphylococcal enterotoxin A (SEA) is an enterotoxin produced mainly by Staphylococcus aureus. In recent years, it has become the most prevalent compound for staphylococcal food poisoning (SFP) around the world. In this study, we isolate new dual-function single-stranded DNA (ssDNA) aptamers by using some new methods, such as the Taguchi method, by focusing on the detection and neutralization of SEA enterotoxin in food and clinical samples. For the asymmetric polymerase chain reaction (PCR) optimization of each round of systematic evolution of ligands by exponential enrichment (SELEX), we use Taguchi L9 orthogonal arrays, and the aptamer mobility shift assay (AMSA) is used for initial evaluation of the protein-DNA interactions on the last SELEX round...
February 26, 2018: Analytical Biochemistry
Boris F Krasnikov
In modern biomedical science, a descriptive study is no longer the major focus of many fields. More researchers are now seeking approaches that will help them obtain maximum information from a single sample or model, which will allow them to make more detailed conclusions than previously about mechanisms that underlie certain phenomena. Clearly, simultaneous measurement of multiple parameters will provide more useful information compared to that which can be assessed through parallel studies with multiple single-parameter measurements...
February 24, 2018: Analytical Biochemistry
Teppo Salminen, Etvi Juntunen, Merja Lahdenranta, Iida Martiskainen, Sheikh M Talha, Kim Pettersson
There is a need for quantitative and sensitive, yet simple point-of-care immunoassays. We have developed a point-of-care microparticle-based immunoassay platform which combines the performance of a microtiter well-based assay with the usability of a rapid assay. The platform contained a separate reaction and detection chambers and microparticles for the solid-phase. Photoluminescent up-converting nanoparticles (UCNPs) were used as labels. The platform was tested with a cardiac troponin I assay, and a limit of detection of 19...
February 24, 2018: Analytical Biochemistry
Pascal Steffen, Christoph Krisp, Wang Yi, Pengyuan Yang, Mark P Molloy, Hartmut Schlueter
Transporting biological samples such as cells or tissues is complicated by the need to maintain integrity and minimise modification and degradation, but this is economically costly as the samples must be shipped in a frozen state. This multi-laboratory study investigated sample variability introduced by non-cooled transport of dried peptide samples for proteomic analysis using mass spectrometry. Human cancer cell tryptic lysates were proteolysed and dried in Australia and shipped by air to Europe and China...
February 24, 2018: Analytical Biochemistry
Jaimie Dufresne, Angelique Florentinus-Mefailoski, Pete Bowden, John G Marshall
The proteins identified from endogenous peptides agreed between serum versus plasma, and tryptic versus non-tryptic peptides, collected by C18 alone and analyzed by liquid chromatography electrospray ionization and tandem mass spectrometry (LC-ESI-MS/MS) including amyloids, apolipoproteins, haptoglobin, complements, fibrinogens, hemopexin, antitrypsin and alpha 2 macroglobulin. Precipitation of polypeptides from plasma in 9 vol of 100% organic solvent followed by stepwise extraction of the insoluble pellet with an increasing fraction of water identified thousands of proteins...
February 24, 2018: Analytical Biochemistry
Jafar Amani, Mehdi Maleki, Alireza Khoshroo, Ali Sobhani-Nasab, Mehdi Rahimi-Nasrabadi
In this work, a label-free electrochemical immunosensor was constructed on the base of poly p-phenylenediamine (PPD) and GR nanocomposite (PPD-GR). Screen-printed electrodes modified with PPD-GR nanocomposite and applied to advance enzyme-free and label free electrochemical immunosensor for detection of protein biomarker neuron-specific enolase (NSE). It was found that the PPD-GR nanocomposite exhibits excellent electrocatalytic activity towards ascorbic acid (AA) oxidation as analytical signal based on EC' mechanism...
February 24, 2018: Analytical Biochemistry
Jonas Johannes Christ, Lars Mathias Blank
Polyacrylamide gel electrophoresis, being the current method of choice for length determination of inorganic polyphosphate (polyP), requires a sequencing apparatus, relies on commercially not available polyP length standards and yields only a chain length distribution. State of the art polyP quantification involves enzymatic hydrolysis of polyP to orthophosphate with the Saccharomyces cerevisiae Exopolyphosphatase 1 (scPpx1p) and subsequent colorimetric orthophosphate detection. Because scPpx1p leaves one pyrophosphate per polyP, short chain polyPs are only partially detected...
February 23, 2018: Analytical Biochemistry
Gaëlle Coussot, Clément Faye, Aurélie Le Postollec, Michel Dobrijevic
The anti-horseradish peroxidase (HRP) antibody is conventionally used in immunohistochemistry. More recently, it has been used as the key element in a gold standard method to evaluate the functionality of antibody-based materials. However, few information are available about its melting temperature and its stability after exposition to laboratory stress conditions including freeze-drying and freeze-thawing cycles. The aim of this study was to evaluate the effects of these environmental constraints on the anti-HRP antibody in order to further use it as a reference in quality control and in the development of new antibody-based materials...
February 22, 2018: Analytical Biochemistry
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