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Analytical Biochemistry

Heather K Le Bleu, Fadia A Kamal, Meghan Kelly, John P Ketz, Michael J Zuscik, Reyad A Elbarbary
Extracting high-quality RNA from articular cartilage is challenging due to low cellularity and high proteoglycan content. This problem hinders efficient application of RNA sequencing (RNA-seq) analysis in studying cartilage homeostasis. Here we developed a method that purifies high-quality RNA directly from cartilage. Our method optimized the collection and homogenization steps so as to minimize RNA degradation, and modified the conventional TRIzol protocol to enhance RNA purity. Cartilage RNA purified using our method has appropriate quality for RNA-seq experiments including an RNA integrity number of ∼8...
November 29, 2016: Analytical Biochemistry
Yu Jin Park, Kyung-Ho Lee, Dong-Myung Kim
We demonstrate the use of a cell-free protein synthesis system as a convenient tool for assessing the relative translational efficiencies of genes. When sfGFP was used as a common reporter gene and co-expressed with a series of target genes, the intensities of sfGFP fluorescence from the co-expression reactions were highly correlated with the individual expression levels of the co-expressed genes. The relative translational efficiencies of genes estimated by this method were reproducible when the same genes were expressed in transformed Escherichia coli, suggesting that this method could be used as a universal tool for prognostic assessment of translational efficiency...
November 28, 2016: Analytical Biochemistry
Ravil R Garafutdinov, Aizilya A Galimova, Assol R Sakhabutdinova
DNA analysis of biological specimens containing degraded nucleic acids such as mortal remains, archaeological artefacts, forensic samples etc. has gained more attention in recent years. DNA extracted from these samples is often inapplicable for conventional polymerase chain reaction (PCR), so for its amplification the nearby primers are commonly used. Here we report the data that clarify the features of PCR with nearby and abutting primers. We have shown that the proximity of primers leads to significant reduction of the reaction time and ensures the successful performance of DNA amplification even in the presence of PCR inhibitors...
November 28, 2016: Analytical Biochemistry
Zahra Shekari, Hamid R Zare, Ali Falahati
The present study aims at the fabrication of a novel electrochemical aptasensor, Ap-GA-AMSN-G, for the label-free determination of hemin and hemoglobin (Hb). Basically, the electrochemical reduction current of hemin or Hb incubated on Ap-GA-AMSN-GCE in the presence of oxygen serves as an excellent signal for quantitative determination of these analytes. By differential pulse voltammetry, the calibration plot was linear in the concentration range of 1.0 × 10(-19)-1.0 × 10(-6) M of hemin and Hb. Also, the detection limits, DL, of hemin and Hb were found to be 7...
November 27, 2016: Analytical Biochemistry
Jian-Dong Zhang, Hua-Lei Wu, Tong Meng, Chao-Feng Zhang, Xiao-Jun Fan, Hong-Hong Chang, Wen-Long Wei
Chiral vicinal amino alcohols are important chiral building blocks and intermediates in the pharmaceutical industry. The transaminase (TAm) catalyzed kinetic resolution of racemic amino alcohols provides a straightforward approach to access these important compounds. This study describes the development of a novel microtiter plate assay to screen vicinal amino alcohol-specific TAms using a tetrazolium red-based colorimetric assay to monitor the rate of α-hydroxy ketone formation at 510 nm. This approach is the first to determine the Michaelis-Menten parameters for a recombinant TAm (PpbauA) from Pseudomonas putida NBRC14164...
November 26, 2016: Analytical Biochemistry
Charles J Richardson, Eric A First
No abstract text is available yet for this article.
November 25, 2016: Analytical Biochemistry
Stefanie Van Wychen, William Long, Stuart K Black, Lieve M L Laurens
A high-throughput and robust application of the 3-methyl-2-benzothiazolinone hydrazone (MBTH) method was developed for carbohydrate determination in microalgae. The standard phenol-sulfuric acid method to quantify carbohydrates is strongly affected by algal biochemical components and exhibits a highly variable response to microalgal monosaccharides. We present a novel use of the MBTH method to accurately quantify carbohydrates in hydrolyzate after acid hydrolysis of algal biomass, without a need for neutralization...
November 24, 2016: Analytical Biochemistry
Dagmar Gelinsky-Wersing, Wolfram Wersing, Wolfgang Pompe
Molecular and functional analysis of small molecule binding to protein can provoke insights into cellular signaling and regulatory systems as well as facilitate pharmaceutical drug discovery. In label free small molecule detection the displacement assay format can be applied. This is beneficial because displacement of high molecular weight receptors is detected instead of low molecular weight ligand as in classical binding analysis. Thus, detection limit is potentially lowered. Using the influenza haemagglutinin (HA) peptide binding to mono or bivalent anti-haemagglutinin peptide antibody displacement assay formats could be established...
November 22, 2016: Analytical Biochemistry
C S Pundir, V Aggarwal
The nanoparticles (NPs) aggregates of lipase from porcine pancreas, glycerol kinase (GK) from Cellulomonas sp. and glycerol-3-phosphate oxidase (GPO) from Aerococcus viridanss were prepared by desolvation and glutaraldehyde crosslinking and functionalized by cysteamine. These enzyme nanoparticles (ENPs) were characterized by transmission electron microscopy (TEM) and Fourier transform infra red (FTIR) spectroscopy. The functionalzed ENPs aggregates were co-immobilized covalently onto polycrystalline Au electrode through thiolated bond...
November 19, 2016: Analytical Biochemistry
Heinz Anderle, Alfred Weber
Trivalent Gd, Tm, and Dy solutions can be used as intrinsic excitation and emission standards to validate the UV and violet-blue wavelength accuracy of a spectrofluorimeter. Europium extends the range into the red. To attain sufficient sensitivity, these luminescent rare earth ions require deuterated reagents or carbonate complexation, which allow the use of ordinary water and thus preparation in virtually any laboratory. Such solutions are particularly valuable as system suitability standards (SST) for protein fluorescence spectroscopy to detect red shifts of the intrinsic fluorescence maximum in stability and storage studies...
November 17, 2016: Analytical Biochemistry
Marianna D Baranovskaya, Victor I Ugarov, Helena V Chetverina, Alexander B Chetverin
The paper reports an inexpensive and efficient procedure for the removal of protein S1 from E. coli ribosomes. It comprises incubation of ribosomes in a pyrimidine polyribonucleotide solution followed by centrifugation of the sample through a sucrose cushion. To avoid co-sedimentation of the S1-bound polypyrimidine with the ribosomes, its length should not exceed several hundred nucleotides. Unlike popular affinity chromatography through a poly(U) Sepharose or poly(U) cellulose column, the method tolerates limited polyribonucleotide degradation by eventual traces of ribonucleases, and can readily be incorporated into standard protocols for the isolation of ribosomes by centrifugation...
November 16, 2016: Analytical Biochemistry
Elodie Desuzinges Mandon, Morgane Agez, Rebecca Pellegrin, Sébastien Igonet, Anass Jawhari
Membrane proteins play crucial role in many cellular processes including cell adhesion, cell-cell communication, signal transduction and transport. To better understand the molecular basis of such central biological machines and in order to specifically study their biological and medical role, it is necessary to extract them from their membrane environment. To do so, it is challenging to find the best solubilization condition. Here we describe, a systematic screening method called BMSS (Biotinylated Membranes Solubilization & Separation) that allow screening 96 conditions at once...
November 12, 2016: Analytical Biochemistry
Ming Zhang, Bo Sun, Qi Zhang, Rong Gao, Qiao Liu, Fangting Dong, Haiqin Fang, Shuangqing Peng, Famei Li, Xianzhong Yan
A quenching, harvesting, and extraction protocol was optimized for cardiomyocytes NMR metabonomics analysis in this study. Trypsin treatment and direct scraping cells in acetonitrile were compared for sample harvesting. The results showed trypsin treatment cause normalized concentration increasing of phosphocholine and metabolites leakage, since the trypsin-induced membrane broken and long term harvesting procedures. Then the intracellular metabolite extraction efficiency of methanol and acetonitrile were compared...
November 11, 2016: Analytical Biochemistry
Aaron J Paredes, Tatiana Naranjo-Palma, Hilda M Alfaro-Valdés, Andrés Barriga, Jorge Babul, Christian A M Wilson
DNA staining in gels has historically been carried out using silver staining and fluorescent dyes like ethidium bromide and SYBR Green I (SGI). Using fluorescent dyes allows recovery of the analyte, but requires instruments such as a transilluminator or fluorimeter to visualize the DNA. Here we described a new and simple method that allows DNA visualization to the naked eye by generating a colored precipitate. It works by soaking the acrylamide or agarose DNA gel in SGI and nitro blue tetrazolium (NBT) solution that, when exposed to sunlight, produces a purple insoluble formazan precipitate that remains in the gel after exposure to light...
November 11, 2016: Analytical Biochemistry
Veronika Rackayova, Cristina Cudalbu, Petra J W Pouwels, Olivier Braissant
Creatine (Cr) is an important organic compound acting as intracellular high-energy phosphate shuttle and in energy storage. While located in most cells where it plays its main roles in energy metabolism and cytoprotection, Cr is highly concentrated in muscle and brain tissues, in which Cr also appears to act in osmoregulation and neurotransmission. This review discusses the basis of Cr metabolism, synthesis and transport within brain cells. The importance of Cr in brain function and the consequences of its impaired metabolism in primary and secondary Cr deficiencies are also discussed...
November 11, 2016: Analytical Biochemistry
Chongxin Xu, Xiaoqin Liu, Cunzheng Zhang, Xiao Zhang, Jianfeng Zhong, Yuan Liu, Xiaodan Hu, Manman Lin, Xianjin Liu
Cry1Ie toxin was an insect-resistant protein used in genetically modified crops (GMC). In this study, a large human VH gene nanobodies phage displayed library was employed to select anti-Cry1Ie toxin antibody by affinity panning. After 5 rounds of panning, total 12 positive monoclonal phage particles were obtained. One of the identified positive phage nanobody was expressed in E.coli BL21 and the purified protein was indicated as a molecular mass of approximately 20 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)...
November 9, 2016: Analytical Biochemistry
Julieta B Carrillo, Diego F Gomez-Casati, Maria V Busi, Mariana Martín
Glucan phosphatases are essential for normal starch degradation in plants and glycogen metabolism in mammals. Here we develop two chromogenic methods for the detection of glucan phosphatase activity in situ after non denaturing poliacrylamide gel electrophoresis; one method uses pNPP and the second one applies BCIP/NBT. The assays are sensitive, fast, simple, reliable and cost-effective preventing the use of radioactive or fluorogenic compounds. Taking advantage of an efficient separation method combined with the reported assays it is possible to obtain information about oligomeric state of the active enzymes as well as to simultaneously detect glucan substrate binding and phosphatase activity...
November 9, 2016: Analytical Biochemistry
Pimchanok Busayapongchai, Sineenat Siri
With increasing concerns of estrogenic effects of endocrine disrupting compounds, the development of simple detection assay for these compounds is an ongoing need. Herein, a simple, rapid, and highly sensitive assay for estradiol (E2) detection was developed using the ligand binding domain of estrogen receptor α (LBD-ERα), the receptor interacting domain of steroid receptor co-activator 1 (RID-SRC1), and gold nanoparticles (AuNPs). The colloidal AuNPs could be stabilized against a salt-induced aggregation by adding LBD-ERα protein...
November 8, 2016: Analytical Biochemistry
Mey Ling Reytor González, Susan Cornell-Kennon, Erik Schaefer, Petr Kuzmič
We propose that the time course of an enzyme reaction following the Michaelis-Menten reaction mechanism can be conveniently described by a newly derived algebraic equation, which includes the Lambert Omega function. Following Northrop's ideas [Anal. Biochem.321, 457-461, 1983], the integrated rate equation contains the Michaelis constant (KM) and the specificity number (kS≡kcat/KM) as adjustable parameters, but not the turnover number kcat. A modification of the usual global-fit approach involves a combinatorial treatment of nominal substrate concentrations being treated as fixed or alternately optimized model parameters...
November 4, 2016: Analytical Biochemistry
Seda Uzunboy, Sema Demirci Çekiç, Ece Eksin, Arzum Erdem, Reşat Apak
An unbalanced excess of oxygen/nitrogen species (ROS/RNS) can give oxidative hazard to DNA and other biomacromolecules under oxidative stress conditions. While the 'comet' assay for measuring DNA damage is neither specific nor practical, monitoring oxidative changes on individual DNA bases and other oxidation products needs highly specialized equipment and operators. Thus, we developed a modified CUPRAC (cupric ion reducing antioxidant capacity) colorimetric method to determine the average total damage on DNA produced by Fenton oxidation, taking advantage of the fact that the degradation products of DNA but not the original macromolecule is CUPRAC-responsive...
November 2, 2016: Analytical Biochemistry
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