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Analytical Biochemistry

Ksenia Maximova, Jakub Wojtczak, Joanna Trylska
Isothermal titration calorimetry (ITC) is a universal technique that directly measures the heat absorbed or released in a process. ITC is typically used to determine thermodynamic parameters of association of molecules without the need to label them. However, ITC is still rarely applied to study chemical reactions catalyzed by enzymes. In addition, these few studies of enzyme kinetic measurements that have been performed were in diluted solutions. Yet, to estimate realistic kinetic parameters, we have to account for the fact that enzymatic reactions in cells occur in a crowded environment because cells contain 200-400 g/L of macromolecular crowders such as proteins, ribosomes and lipids...
November 12, 2018: Analytical Biochemistry
Malik Saddam Khan, Wenjuan Zhu, Asghar Ali, Shah Masood Ahmad, Xiaojian Li, Lei Yang, Yaoguang Wang, Huan Wang, Qin Wei
A novel label-free electrochemiluminescent (ECL) immunosensor based upon luminol functionalized platinum nanoparticles loaded on graphene sheets (Lu-Pt@GS) as sensing platform was fabricated for highly sensitive and selective determination of prostate specific antigen (PSA). In this work, for the first time luminol was employed as both ECL luminescence reagent and reductants to in-situ reduce H2 PtCl6 forming Pt NPs on surface of GS. A great deal of luminol could be attached onto the surface of Pt NPs within the reduction process, which can generate strong ECL emission...
November 12, 2018: Analytical Biochemistry
Cunjie Hu, Jianping Li, Liqiang Yan
An excellent fluorescent probe with "turn on" phenomenon for sensitive monitoring hypochlorite (ClO- ) was prepared using the mild condensation reaction between coumarin and hydroxylamine (NH2 OH). The probe possessed potent selectivity to hypochlorite (ClO- ) with obvious color changes from yellow to light yellow and green to blue fluorescence emission, which could be noticed by the naked eye. Moreover, the probe has been succeeded in imaging ClO- in living A549 cells and thus has the potential prospect in the visual detection of intracellular ClO- ...
November 12, 2018: Analytical Biochemistry
Linxi Hou, Mingqin Shangguan, Zhen Lu, Sili Yi, Xingzong Jiang, Heng Jiang
A new cyclometalated iridium(III) complex-based fluorescence probe (IrCN) for hypochlorite (ClO- ) has been synthesized and characterized. The probe displayed nonfluorescent around 577 nm, while a 54-fold enhancement in fluorescence emission intensity was observed after the addition of ClO- due to the removal of C=N isomerization effect. Such "turn-on" fluorescence probe worked excellently in wide pH range (5-12) with short response time (<20 s) and the detection limit was as low as 0.11 μM...
November 11, 2018: Analytical Biochemistry
Maria Khihon Rokhas, Johanna Liljestrand Rönn, Christer Wiklund, Åsa Emmer
A method for analysis of proteins from spermatophores transferred from male to female Pieris napi butterflies during mating has been developed. The proteins were solubilized from the dissected spermatophores using different solubilization agents (water, methanol, acetonitrile and hexafluoroisopropanol). Capillary electrophoresis (CE) analysis was performed using an acidic background electrolyte containing a fluorosurfactant to avoid protein-wall adsorption, and to increase separation performance. The samples were also analyzed with matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS), in a lower m/z range (1000-6000) and a higher m/z range (6000-12000)...
November 10, 2018: Analytical Biochemistry
Sujatha Chilakala, Iteen Cheng, Ireen Lee, Yan Xu
A method is proposed in this paper for the determination of oxygen-18 labeled phosphate so that positional isotope experiments using sensitive and rapid liquid chromatography-QTOF-mass spectrometry (LC-QTOF-MS) experiments can be carried out. The positional isotope exchange technique is a useful tool in understanding the mechanisms and kinetics of many enzyme-catalyzed reactions. Detection of the positions and concentration of these exchanged isotopes is the key. Gas chromatography-mass spectrometry (GC-MS) and nuclear magnetic resonance imaging are commonly used analytical techniques for measurement of 18 O/16 O, 31 P and 15 N isotope enrichment...
November 9, 2018: Analytical Biochemistry
Liang Qiao, Dongqing Xie
Accurately targeting metal ion-binding sites solely from protein sequences is valuable for both basic experimental biology and drug discovery studies. Although considerable progress has been made, metal ion-binding site prediction is still a challenging problem due to the small size and high versatility of the metal ions. In this paper, we develop a ligand-specific predictor called MIonSite for predicting metal ion-binding sites from protein sequences. MIonSite first employs protein evolutionary information, predicted secondary structure, predicted solvent accessibility, and conservation information calculated by Jensen-Shannon Divergence score to extract the discriminative feature of each residue...
November 8, 2018: Analytical Biochemistry
F Yao, X Y Xu, Q Pan
Plasmids of Lactobacillus plantarum PC518 cannot be effectively extracted by existing methods. It was studied that the effect of lysozyme treatment and removal on plasmid extraction by 7 protocols. The modified method was compared with a commercial kit using L. plantarum PC518, 410, 9L15, and JS193 and Weissella cibaria M2 as the tested strains. The results suggested that the step of lysozyme removal is the key to improve the efficiency of plasmid extraction. The concentrations of plasmid DNA isolated from the 5 tested strains were increased by 10...
November 5, 2018: Analytical Biochemistry
Kelly J Goodwin, Eric Gangl, Ujjal Sarkar, Petar Pop-Damkov, Natalie Jones, Alexandra Borodovsky, Richard Woessner, Adrian J Fretland
Adenosine is known to be an important signaling molecule in many physiological processes and has recently been shown to be an important molecule in oncology. A fit for purpose method has been developed for the quantification of adenosine in murine tumor samples using pre-column derivatization and liquid chromatography-mass spectrometry (LC-MS/MS). To overcome adenosine quantification challenges, derivatization with dansyl chloride was employed. This derivatization technique, following protein precipitation and liquid-liquid extraction, improved the sensitivity and selectivity of adenosine in tumor samples through the reduction of endogenous interference and matrix effects...
November 5, 2018: Analytical Biochemistry
Takayuki Saitoh, Yasumitsu Seto, Yukichi Fujikawa, Noriaki Iijima
We report here a liquid chromatography/electrospray ionization-tandem mass spectrometry assay for the quantification of three isoforms of antimicrobial peptide (AMP), chrysophsin-1, -2 and -3, in the red sea bream, Pagrus (Chrysophrys) major. Chrysophsin-1 was mainly distributed in the pyloric caeca and gills, followed by intestine and stomach. Chrysophsin-2 was detected in the gills and stomach, but chrysophsin-3 was only in the gills. The present procedure is valuable as a general method for simultaneous determination of the level of multiple AMP isoforms in fish tissues, and the data give important information for understanding the significance of each AMP isoform in host defense...
November 4, 2018: Analytical Biochemistry
Shereen A Murugayah, Suzanne L Warring, Monica L Gerth
N-acyl-l-homoserine lactone (AHL) acylases are a well-known group of enzymes that disrupt quorum sensing in Gram-negative bacteria by degrading AHL signalling molecules. This degradation of signalling molecules (termed 'quorum quenching') has potential uses in the prevention or reduction of biofilm formation and/or bacterial infections. Therefore, there is a great deal of interest in the identification and characterisation of quorum quenching enzymes. Here, we present an optimised fluorescamine-based assay for the detection of AHL acylase activity and demonstrate it can be used in a high-throughput screening format...
November 2, 2018: Analytical Biochemistry
Lauana Fogaça de Almeida, Leonardo Nazário de Moraes, Lucilene Dos Santos Delazari, Guilherme Targino Valente
Mass spectrometry is the most used method for protein identification and quantification. Here we developed four protein extraction protocols precisely for mass spectrometry, and we compared with other ones already published. The best protocol developed by us consists on a simple extraction solution, a heat-shock step, and does not use protease inhibitor; moreover, it is the most efficient and uniform among replicates, besides to be safe, cheap and fast. That method also provided the highest number of proteins uniquely identified and allows finding a diversity of protein classes, which their absence is a problem to be avoided...
November 1, 2018: Analytical Biochemistry
Victoria Medawar-Aguilar, Claudio F Jofre, Martín A Fernández-Baldo, Andrés Alonso, Sergio Angel, Julio Raba, Sirley V Pereira, Germán A Messina
This article describes a microfluidic LIF immunosensor for the quantitative determination of anti-Toxoplasma gondii IgG (anti-T. gondii) specific antibodies. The serological detection of these antibodies plays a crucial role in the clinical diagnosis of toxoplasmosis. Zinc oxide nanoparticles (ZnO-NPs) obtained by wet chemical procedure were covered with chitosan and then used to conjugate T-gondii antigens into the central microfluidic channel. Serum samples containing anti-T-gondii IgG antibodies were injected into the immunosensor where they interact immunologically with T...
October 28, 2018: Analytical Biochemistry
Muhammad Kabir, Muhammad Arif, Farman Ali, Saeed Ahmad, Zar Nawab Khan Swati, Dong-Jun Yu
Membrane protein is a pivotal constituent of a cell that exerts a crucial influence on diverse biological processes. The accurate identification of membrane protein types is deeply essential for revealing molecular mechanisms and drug development. Primarily, several traditional methods were exploited to classify these types. However, experimental methods are laborious, time-consuming, and costly due to rapid exploration of uncharacterized protein sequences generated in the postgenomic era. Hence, machine learning-based methods are more indispensable for reliable and fast identification of membrane protein types...
October 27, 2018: Analytical Biochemistry
Mark L Sowers, Jason Herring, William Zhang, Hui Tang, Yang Ou, Wei Gu, Kangling Zhang
A major hallmark of cancer is a perturbed metabolism resulting in high demand for various metabolites, glucose being the most well studied. While glucose can be converted into pyruvate for ATP production, the serine synthesis pathway (SSP) can divert glucose to generate serine, glycine, and methionine. In the process, the carbon unit from serine is incorporated into the one-carbon pool which makes methionine and maintains S-adenosylmethionine levels, which are needed to maintain the epigenetic landscape and ultimately controlling what genes are available for transcription...
October 26, 2018: Analytical Biochemistry
Deepsikha Srivastava, Gosala Raja Kukkuta Sarma, Delon Snehal Dsouza, Monita Muralidharan, Krishnamachari Srinivasan, Amit Kumar Mandal
Reduction of a disulfide linkage between cysteine residues in proteins, a standard step in the preanalytical preparation of samples in conventional proteomics approach, presents a challenge to characterize S-glutathionylation of proteins. S-glutathionylation of proteins has been reported in medical conditions associated with high oxidative stress. In the present study, we attempted to characterize glutathionylation of CSF proteins in patients with multiple sclerosis which is associated with high oxidative stress...
October 24, 2018: Analytical Biochemistry
Jing Wen, Lina Yang, Feng Qin, Longshan Zhao, Zhili Xiong
A UHPLC-MS/MS untargeted serum metabonomic method combined with quantitative analysis of five potential biomarkers in rat serum was developed and validated, to further understand the anti-liver injury effect of Si-Ni-San and its mechanism on liver injury rats in this study. The metabolites were separated and identified on BEH C18 column (100 mm × 2.1 mm, 1.7 μm) using the ACQUITY UHPLC-MS system (Waters Corp., Milford, MA, USA). Principal component analysis (PCA) was used to identify potential biomarkers...
October 24, 2018: Analytical Biochemistry
Junhyun Chong, Hayun Chong, Ji Hoon Lee
An aptasensor with guanine chemiluminescence detection was developed for the early diagnosis of prostate cancer with the simultaneous quantifications of prostate specific antigen (PSA) and vascular endothelial growth factor (VEGF). A PSA DNA aptamer conjugated with Texas Red emits red light in guanine chemiluminescence reaction, whereas a VEGF DNA aptamer conjugated with 6-FAM emits green light. The PSA and VEGF aptamers immobilized on the surface of paramagnetic beads mixed with a sample were incubated for 30 min...
October 24, 2018: Analytical Biochemistry
Kelly M Hanson, Jacob N Finkelstein
We present a real-time, high-throughput, and cost-effective method of detecting apoptosis in vitro using a previously developed reagent that detects caspase activation by fluorescence. Current methods of assessing apoptosis fail to account for the dimension of time, and thus are limited in data yielded per sample. This reagent allows real-time detection of apoptosis, but until now has been restricted to a costly automated detection system. Here, we describe apoptosis detection with the Essen Bioscience IncuCyte® Caspase-3/7 Reagent using a multimode microplate reader, a common instrument in biological laboratories, which may be used prior to or in lieu of the automated system...
October 24, 2018: Analytical Biochemistry
Pooja Kumari, Surabhi Lavania, Shaifali Tyagi, Abhijeet Dhiman, Deepak Rath, Divya Anthwal, Rakesh Kumar Gupta, Neera Sharma, A K Gadpayle, R S Taneja, Lokesh Sharma, Yusra Ahmad, Tarun Kumar Sharma, Sagarika Haldar, Jaya Sivaswami Tyagi
Pleural tuberculosis (pTB) is diagnosed by using a composite reference standard (CRS) since microbiological methods are grossly inadequate and an accurate diagnostic test remains an unmet need. The present study aimed to evaluate the utility of Mycobacterium tuberculosis (Mtb) antigen and DNA-based tests for pTB diagnosis. Patients were classified as 'Definite TB', 'Probable TB' and 'Non-TB' disease according to the CRS. We assessed the performance of in-house antigen detection assays, namely antibody-based Enzyme-Linked ImmunoSorbent Assay (ELISA) and aptamer-based Aptamer-Linked Immobilized Sorbent Assay (ALISA), targeting Mtb HspX protein and DNA-based tests namely, Xpert MTB/RIF and in-house devR-qPCR...
October 22, 2018: Analytical Biochemistry
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