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Analytical Biochemistry

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https://www.readbyqxmd.com/read/28624296/a-refined-dna-methylation-detection-method-using-mspji-coupled-quantitative-pcr
#1
Christopher J Petell, Gilbert Loiseau, Ryan Gandy, Sriharsa Pradhan, Humaira Gowher
DNA methylation is a highly conserved epigenetic modification with critical roles ranging from protection against phage infection in bacteria to the regulation of gene expression in mammals. DNA methylation at specific sequences can be measured by using methylation dependent or sensitive restriction enzymes coupled to semi- or quantitative PCR (MD-qPCR). This study reports a refined MD-qPCR method for detecting gain or loss of DNA methylation at specific sites through the specific use of MspJI or HpaII, respectively...
June 15, 2017: Analytical Biochemistry
https://www.readbyqxmd.com/read/28624297/aptasensors-for-quantitative-detection-of-salmonella-typhimurium
#2
REVIEW
Najmeh Ansari, Zhouping Wang, Rezvan Yazdian-Robati, Mahin Shahdordizadeh, Kiarash Ghazvini
Salmonella is one of the most frequent causes of food borne infectious disease. Among nearly 2500 documented serotypes are reported, Salmonella Typhimurium is the number one serotype associated with salmonellosis worldwide. Many different methods have been developed for the detection and quantification of S. typhimurium. Most of these assays are usually expensive, time consuming and require difficult sample preparation steps. Therefore, it is necessary to develop rapid, robust, cost-effective and sensitive alternative detection methods...
June 14, 2017: Analytical Biochemistry
https://www.readbyqxmd.com/read/28623075/thioflavin-t-fluorescence-to-analyse-amyloid-formation-kinetics-measurement-frequency-as-a-factor-explaining-irreproducibility
#3
Mathew Sebastiao, Noe Quittot, Steve Bourgault
The most frequent method to monitor amyloid formation relies on the fluorescence of thioflavin T (ThT). The present study reports a novel factor of irreproducibility in ThT kinetic assays performed in microplate. Discrepancies among kinetics of amyloid assembly, performed under quiescent conditions, were associated with the frequency of fluorescence measurement. Evaluating self-assembly of the islet amyloid polypeptide at short intervals hastened its fibrillization. This observation was confirmed by transmission electron microscopy, circular dichroism spectroscopy and 8-anilino-1-naphthalenesulfonic acid fluorescence...
June 13, 2017: Analytical Biochemistry
https://www.readbyqxmd.com/read/28610874/on-nylon-membrane-detection-of-nucleic-acid-molecules-by-rolling-circle-amplification
#4
Xinhui Xu, Beibei Zhang, Ping Gan, Jian Wu, Wei Dai, Ling Zhang, Jinke Wang
Positively-charged nylon membrane (NM) is a general solid-phase support for nucleic acid detection due to its convenient immobilization of nucleic acid materials by direct electrostatic adherence and simple UV crosslinking. Rolling circle amplification (RCA) is a widely used isothermal DNA amplification technique for nucleic acid detection. Near-infrared fluorescence (NIRF) is a new fluorescence technique with high sensitivity due to low background. This study developed a simple method for detecting nucleic acid molecules by combining the advantages of NM, RCA and NIRF, named NIRF-based solid phase RCA on nylon membrane (NM-NIRF-sRCA)...
June 10, 2017: Analytical Biochemistry
https://www.readbyqxmd.com/read/28606426/electrochemical-label-free-and-sensitive-nanobiosensing-of-dna-hybridization-by-graphene-oxide-modified-pencil-graphite-electrode
#5
F Ahour, A Shamsi
Based on the strong interaction between single-stranded DNA (ss-DNA) and graphene material, we have constructed a novel label-free electrochemical biosensor for rapid and facile detection of short sequences ss-DNA molecules related to hepatitis C virus 1a using graphene oxide modified pencil graphite electrode. The sensing mechanism is based on the superior adsorption of single-stranded DNA to GO over double stranded DNA (ds-DNA). The intrinsic guanine oxidation signal measured by differential pulse voltammetry (DPV) has been used for duplex DNA formation detection...
June 9, 2017: Analytical Biochemistry
https://www.readbyqxmd.com/read/28602750/single-molecule-raman-spectroscopic-assay-to-detect-transgene-from-gm-plants
#6
Ulhas S Kadam, Rahul L Chavhan, Burkhard Schulz, Joseph Irudayaraj
Substantial concerns have been raised for the safety of transgenics on human health and environment. Many organizations, consumer groups, and environmental agencies advocate for stringent regulations to avoid transgene products' contamination in food cycle or in nature. Here we demonstrate a novel approach using surface enhanced Raman spectroscopy (SERS) to detect and quantify transgene from GM plants. We show a highly sensitive and accurate quantification of transgene DNA from multiple transgenic lines of Arabidopsis...
June 8, 2017: Analytical Biochemistry
https://www.readbyqxmd.com/read/28600127/detection-and-purification-of-backbone-cyclized-proteins-using-a-bacterially-expressed-anti-myc-tag-single-chain-antibody
#7
Sushma Chauhan, Chen Yuan Hou, Sang Taek Jung, Taek Jin Kang
A myc-tag and of which recognition by an antibody 9E10 has long been used for the detection and purification of recombinant proteins. We have previously expanded the application of the tag to the specific detection and purification of backbone-cyclized proteins. Here we sought a more practical way to using the 9E10 antibody by expressing its single chain antibody (scAb) form in Escherichia coli. The combined use of a strong T7 promoter and auto-induction strategy rather than early to mid-log induction of a Lac promoter resulted in the soluble over-expression of 9E10 scAb...
June 7, 2017: Analytical Biochemistry
https://www.readbyqxmd.com/read/28595966/characterization-of-pkac%C3%AE-enzyme-kinetics-and-inhibition-in-an-hplc-assay-with-a-chromophoric-substrate
#8
Nicole M Luzi, Charles E Lyons, Darrell L Peterson, Keith C Ellis
Here we describe a convenient, inexpensive, and non-hazardous method for the measurement of the kinase activity of the catalytic subunit of cAMP-dependent protein kinase (PKACα). The assay is based on the separation of a substrate peptide labeled with a strong chromophore from the phosphorylated product peptide by high-performance liquid chromatograph (HPLC) and quantification of the product ratiometrically at a wavelength in the visual spectrum (Vis). The utility and reliability of the HPLC-Vis assay were demonstrated by characterizing the kinetic parameters (KM, Vmax) of the new Rh-MAB-Kemptide substrate, a commercially prepared TAMRA-Kemptide substrate, and ATP as well as the potency (IC50, Ki) of the known PKACα inhibitors H89 and PKI(5-24)...
June 5, 2017: Analytical Biochemistry
https://www.readbyqxmd.com/read/28587811/dansylglycine-a-fluorescent-probe-for-specific-determination-of-halogenating-activity-of-myeloperoxidase-and-eosinophil-peroxidase
#9
Luiza de Carvalho Bertozo, Maria Luiza Zeraik, Valdecir Farias Ximenes
Myeloperoxidase (MPO) and eosinophil peroxidase (EPO) are enzymes present in neutrophil and eosinophil leukocytes, respectively. Here, we present the development of a sensitive and specific assay for determination of the halogenating enzymatic activity of MPO and EPO based on the electrophilic attack of HOCl and HOBr on aromatic ring of dansylglycine (DG). We found that the intrinsic fluorescence of DG was promptly depleted by the action of these acids. In the presence of the enzymes, the fluorescence bleaching was dependent of chloride (Cl(-)) and bromide (Br(-)), which makes the assay able to distinguish the halogenating from the peroxidase activity...
June 3, 2017: Analytical Biochemistry
https://www.readbyqxmd.com/read/28579488/effective-cell-free-drug-screening-protocol-for-protein-protein-interaction
#10
Shaked Ashkenazi, Alexander Plotnikov, Anat Bahat, Rivka Dikstein
Specific protein-protein interaction (PPI) is an essential feature of many cellular processes however, targeting these interactions by small molecules is highly challenging due to the nature of the interaction interface. Thus, screening for PPI inhibitors requires enormous number of compounds. Here we describe a simple and improved protocol designed for a search of direct PPI inhibitors. We engineered a bacterial expression system for the split-Renilla luciferase (RL) complementation assay that monitors PPI...
June 1, 2017: Analytical Biochemistry
https://www.readbyqxmd.com/read/28577993/fluorescence-detection-of-the-transglycosylation-activity-of-amylosucrase
#11
Dong-Ho Seo, Jong-Hyun Jung, Cheon-Seok Park
The purpose of this study was to investigate the novel fluorescence-based assay for the transglycosylation activity of amylosucrase (ASase). The transglycosylation activity of ASase from Deinococcus geothermalis (DGAS), ASase from Neisseria polysaccharea (NPAS), and DGAS-B (chimeric ASase wherein the B domain from DGAS was exchanged with the B domain of NPAS in a DGAS background) was applied to modify 4-methlylumberlliferone (MU) to 4-methylumberlliferone glucoside (MUG) using MU as an acceptor and sucrose as a glucoside donor...
May 31, 2017: Analytical Biochemistry
https://www.readbyqxmd.com/read/28576440/photometric-assay-of-maltose-and-maltose-forming-enzyme-activity-by-using-4-alpha-glucanotransferase-dpe2-from-higher-plants
#12
Julia Smirnova, Alisdair R Fernie, Christian C M Spahn, Martin Steup
Maltose frequently occurs as intermediate of the central carbon metabolism of prokaryotic and eukaryotic cells. Various mutants possess elevated maltose levels. Maltose exists as two anomers, (α- and β-form) which are rapidly interconverted without requiring enzyme-mediated catalysis. As maltose is often abundant together with other oligoglucans, selective quantification is essential. In this communication, we present a photometric maltose assay using 4-alpha-glucanotransferase (AtDPE2) from Arabidopsis thaliana...
May 31, 2017: Analytical Biochemistry
https://www.readbyqxmd.com/read/28571750/measuring-o-glcnac-cleavage-by-oga-and-cell-lysates-on-a-peptide-microarray
#13
Suhela Sharif, Jie Shi, Mostafa Bourakba, Rob Ruijtenbeek, Roland J Pieters
O-GlcNAcylation is a post-translational modification resulting from the addition of an N-acetylglucosamine moiety to the hydroxyl groups of serine and threonine residues of nuclear and cytoplasmic proteins. In addition, O-GlcNAcylated proteins can be phosphorylated, which suggests the possibility for crosstalk between O-GlcNAcylation and phosphorylation. Dysregulation of O-GlcNAcylation affects cell signaling, transcriptional regulation, cell cycle control and can e.g. lead to tumorigenesis and tumor metastasis...
May 29, 2017: Analytical Biochemistry
https://www.readbyqxmd.com/read/28552759/generation-of-smurf2-knockout-human-cells-using-the-crispr-cas9-system
#14
Dhanoop Manikoth Ayyathan, Nataša Ilić, Hava Gil-Henn, Michael Blank
The HECT domain E3 ubiquitin ligase SMURF2 regulates stability of several key protein targets involved in tumorigenesis, cell proliferation, migration, differentiation, and senescence. While altered levels and aberrant cellular distribution of SMURF2 were reported in different types of cancer, its role in tumorigenesis is far from understood. To elucidate the role of SMURF2 in cancer, appropriate human cancer cell models are needed. Here, we describe approaches that can be used to generate human normal and cancer cell strains knocked-out for SMURF2 using the clustered regularly interspaced short palindromic repeats (CRISPR/Cas9) gene-editing technology...
May 26, 2017: Analytical Biochemistry
https://www.readbyqxmd.com/read/28552758/fluorescence-polarization-based-competition-binding-assay-for-c-jun-n-terminal-kinases-1-and-2
#15
Francesco Ansideri, Marcel Dammann, Frank M Boeckler, Pierre Koch
In order to evaluate the isoform selectivity of novel inhibitors within the c-Jun N-terminal kinase (JNK) family, a fluorescence polarization-based competition binding assay, previously developed for JNK3, was extended to the other isoforms JNK1 and JNK2. The assay is based on the displacement of a versatile fluorescent pyridinylimidazole-based probe and was validated by testing the precursor of the probe as well as standard JNK inhibitors.
May 25, 2017: Analytical Biochemistry
https://www.readbyqxmd.com/read/28552757/selective-fluorescence-detection-method-for-selenide-and-selenol-using-monochlorobimane
#16
Takeshi Imai, Tatsuo Kurihara, Nobuyoshi Esaki, Hisaaki Mihara
The low redox potential of selenide and selenol is physiologically important, as it confers efficient catalytic abilities to selenoproteins. Quantitative determination of selenol and selenide provide important clues for understanding the metabolism and physiological function of selenium. However, selective detection of selenol and selenide is extremely difficult because of their chemical similarity to thiol and sulfide. In this study, we established a highly sensitive, selective, quantitative, and simple method for detection of selenol and selenide, using a reaction with monochlorobimane (MCB), followed by ethyl acetate extraction of the product syn-(methyl,methyl)bimane...
May 25, 2017: Analytical Biochemistry
https://www.readbyqxmd.com/read/28552756/dopamine-modified-hyperbranched-tio2-arrays-based-ultrasensitive-photoelectrochemical-immunosensor-for-detecting-neuron-specific-enolase
#17
He Li, Qiyou Xiao, Jiaxin Lv, Qin Lei, Yujie Huang
In this work, three-dimensional (3D) hyperbranched TiO2 nanorod arrays were synthesized and used to fabricate dopamine sensitized photoelectrochemical (PEC) biosensor. To increase the lifetime of charge carriers and enhance the photocurrent responses signal, a delicate signal amplification strategy by introducing dopamine (DA) as sensitizer was developed. The dopamine sensitized TiO2 can shorten the carrier diffusion distance, improve light harvesting efficiency and charge collection efficiency, which results in performance improvement of the as-obtained PEC sensor...
May 25, 2017: Analytical Biochemistry
https://www.readbyqxmd.com/read/28545867/the-photostability-of-the-commonly-used-biotin-4-fluorescein-probe
#18
Richard A Haack, Kerry M Swift, Qiaoqiao Ruan, Richard J Himmelsbach, Sergey Y Tetin
Biotin-4-fluorescein (B4F) is a commonly used fluorescent probe for studying biotin-(strept)avidin interactions. During a characterization study of an anti-biotin antibody, using B4F as the probe, we noticed a discrepancy in the expected and experimentally determined number of biotin binding sites. Analytical testing showed that the biotin moiety in the probe undergoes a photosensitized oxidation to produce a mixture of biotin sulfoxides which has the potential to impact the quantitation of binding sites using this fluorescent probe...
May 22, 2017: Analytical Biochemistry
https://www.readbyqxmd.com/read/28545866/isolation-of-single-cells-for-protein-therapeutics-using-microwell-selection-and-surface-plasmon-resonance-imaging
#19
F Abali, M Stevens, A G J Tibbe, L W M M Terstappen, P N van der Velde, R B M Schasfoort
Here the feasibility is demonstrated that by combining Surface Plasmon Resonance Imaging (SPRi) and self-sorting microwell technology product secretion of individual cells can be monitored. Additionally isolation of the selected cells can be performed by punching the cells from the microwells using coordinates of the positions of microwells obtained with SPRi. Cells of interest can be retrieved sterile from the microwell array for further cultivation.
May 22, 2017: Analytical Biochemistry
https://www.readbyqxmd.com/read/28529050/an-optical-alignment-system-improves-precision-of-soluble-aggregate-quantitation-by-sedimentation-velocity-analytical-ultracentrifugation
#20
Brandon L Doyle, Ivan L Budyak, Adam P Rauk, William F Weiss
Appropriate characterization of soluble aggregates is an important aspect of biologics development and manufacturing, and sedimentation velocity analytical ultracentrifugation (SV-AUC) is often used an orthogonal technique to size-exclusion chromatography (SEC) for this purpose. Precise quantification of low levels of soluble aggregates by SV-AUC can be adversely impacted by improper cell alignment. This report describes the development of an optical system capable of quantifying cell alignment that affords a substantial improvement compared to historical approaches...
May 19, 2017: Analytical Biochemistry
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