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Covalent labeling

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https://www.readbyqxmd.com/read/27934217/fluorescence-and-energy-transfer-in-dye-labeled-dna-crystals
#1
Joseph S Melinger, Ruojie Sha, Chengde Mao, Nadrian C Seeman, Mario G Ancona
DNA crystals make it possible to organize guest molecules into specific periodic 3D patterns at the nanoscale, and thereby to create novel macroscopic objects with potentially useful functionality. Here, we describe the fluorescence and energy transfer properties of DNA crystals that are self-assembled from DNA tensegrity triangles with covalently attached Cy3 and Cy5 dyes. When compared to reference DNA strands in solution, the fluorescence measurements indicate that the dyes in the crystal experience a more homogeneous environment, resulting in a 2-fold increase in Cy3 quantum yield and single-exponential Cy3 fluorescence decays...
December 8, 2016: Journal of Physical Chemistry. B
https://www.readbyqxmd.com/read/27933951/identification-of-the-binding-site-of-chroman-4-one-based-sirtuin-2-selective-inhibitors-using-photoaffinity-labeling-in-combination-with-tandem-mass-spectrometry
#2
Tina Seifert, Marcus Malo, Johan Lengqvist, Carina Sihlbom, Elina M Jarho, Kristina Luthman
Photoaffinity labeling (PAL) was used to identify the binding site of chroman-4-one-based SIRT2-selective inhibitors. The photoactive diazirine 4, a potent SIRT2 inhibitor, was subjected to detailed photochemical characterization. In PAL experiments with SIRT2, a tryptic peptide originating from the covalent attachment of photoactivated 4 was identified. The peptide covers both the active site of SIRT2 and the proposed binding site of chroman-4-one-based inhibitors. A high-power LED was used as source for the monochromatic UV light enabling rapid photoactivation...
December 8, 2016: Journal of Medicinal Chemistry
https://www.readbyqxmd.com/read/27931005/microfluidic-immunosensor-for-rapid-and-highly-sensitive-salivary-cortisol-quantification
#3
V Pinto, P Sousa, S O Catarino, M Correia-Neves, G Minas
This paper presents a novel poly(dimethylsiloxane) (PDMS) microfluidic immunosensor that integrates a complementary metal-oxide-semiconductor (CMOS) optical detection system for a rapid and highly-sensitive quantification of salivary cortisol. The simple and non-invasive method of saliva sampling provides an interesting alternative to the blood, allowing a fast sampling at short intervals, relevant for many clinical diagnostic applications. The developed approach is based on the covalent immobilization of a coating antibody (Ab), a polyclonal anti-IgG, onto a treated PDMS surface...
November 30, 2016: Biosensors & Bioelectronics
https://www.readbyqxmd.com/read/27929747/localized-conformational-interrogation-of-antibody-and-antibody-drug-conjugates-by-site-specific-carboxyl-group-footprinting
#4
Lucy Yan Pan, Oscar Salas-Solano, John F Valliere-Douglass
Establishing and maintaining conformational integrity of monoclonal antibodies (mAbs) and antibody-drug conjugates (ADCs) during development and manufacturing is critical for ensuring their clinical efficacy. As presented here, we applied site-specific carboxyl group footprinting (CGF) for localized conformational interrogation of mAbs. The approach relies on covalent labeling that introduces glycine ethyl ester tags onto solvent-accessible side chains of protein carboxylates. Peptide mapping is used to monitor the labeling kinetics of carboxyl residues and the labeling kinetics reflects the conformation or solvent-accessibility of side chains...
December 8, 2016: MAbs
https://www.readbyqxmd.com/read/27929154/a-platform-with-connections-in-many-directions-further-remarkable-facets-to-the-multifaceted-methylbiquinoxen-dication
#5
Nicolas Leblanc, Damiano Genovese, Luisa De Cola, Annie K Powell
The N,N'-dimethyl-3,3'-biquinoxalinium "methylbiquinoxen" dicationic platform is revealed to have even more fascinating possibilities than we originally thought in terms of its chemical versatility. In addition to its rich redox chemistry and coordination abilities, we have now unveiled an unexpected Lewis acid/base chemistry linked with a tuneable switching of its luminescence properties. This, amongst other things, allows for the facile fluorescent covalent labelling of hydroxyl-terminated materials. This platform provides intriguing chemical prospects realised in molecular systems such as porphyrins as well as an easy alternative functionalisation methodology to that provided by click-chemistry...
December 8, 2016: Physical Chemistry Chemical Physics: PCCP
https://www.readbyqxmd.com/read/27925692/a-method-to-generate-and-analyze-modified-myristoylated-proteins
#6
Huanyao Gao, Wei Sun, Zhiquan Song, Yanbao Yu, Li Wang, Xian Chen, Qisheng Zhang
Covalent lipid modification of proteins is essential to their cellular localizations and functions. Engineered lipid motif coupled with bio-orthogonal chemistry has been utilized to identify myristoylated or palmitoylated proteins in cells. However, whether modified proteins have similar properties as endogenous ones has not been well investigated mainly due to lack of methods to generate and analyze purified proteins. We have developed a method that utilizes metabolic interference and mass spectrometry to produce and analyze modified, myristoylated small GTPase ADP-ribosylation factor 1 (Arf1)...
December 7, 2016: Chembiochem: a European Journal of Chemical Biology
https://www.readbyqxmd.com/read/27925666/enriching-s-4-u-rna-using-methane-thiosulfonate-mts-chemistry
#7
Erin E Duffy, Matthew D Simon
Metabolic labeling of cellular RNA is a useful approach to study RNA biology. 4-Thiouridine (s(4) U) is a convenient nucleoside for metabolic labeling because it is cell permeable and is incorporated into newly transcribed RNA, and the sulfur moiety provides a handle for biochemical purification. However, a critical step in the purification of s(4) U-RNA is the efficiency of the chemistry used to enrich s(4) U-RNA. Here, we present a protocol for s(4) U-RNA enrichment that includes efficient and reversible covalent chemistry to biotinylate s(4) U-RNA using the activated disulfide methane thiosulfonate conjugated to biotin (MTS-biotin), followed by enrichment on streptavidin beads...
December 7, 2016: Current Protocols in Chemical Biology
https://www.readbyqxmd.com/read/27923620/fluorescent-diphenylphosphonate-based-probes-for-detection-of-serine-protease-activity-during-inflammation
#8
Laura E Edgington-Mitchell, Nicholas Barlow, Luigi Aurelio, Aminath Samha, Monika Szabo, Bim Graham, Nigel Bunnett
Activity-based probes are small molecules that covalently bind to the active site of a protease in an activity-dependent manner. We synthesized and characterized two fluorescent activity-based probes that target serine proteases with trypsin-like or elastase-like activity. We assessed the selectivity and potency of these probes against recombinant enzymes and demonstrated that while they are efficacious at labeling active proteases in complex protein mixtures in vitro, they are less valuable for in vivo studies...
November 25, 2016: Bioorganic & Medicinal Chemistry Letters
https://www.readbyqxmd.com/read/27919613/cellular-uptake-of-glucoheptoamidated-poly-amidoamine-pamam-g3-dendrimer-with-amide-conjugated-biotin-a-potential-carrier-of-anticancer-drugs
#9
Łukasz Uram, Magdalena Szuster, Aleksandra Filipowicz, Magdalena Zaręba, Elżbieta Wałajtys-Rode, Stanisław Wołowiec
In search for soluble derivatives of PAMAM dendrimers as potential carriers for hydrophobic drugs, the conjugates of PAMAM G3 with biotin, further converted into glycodendrimer with d-glucoheptono-1,4-lactone, were prepared. Polyamidoamine dendrimer (PAMAM) of third generation, G3 was functionalized with four biotin equivalents covalently attached to terminal amine nitrogens via amide bond G3(4B). The remaining 28 amine groups were blocked by glucoheptoamide substituents (gh) to give G3(4B28gh) or with one fluorescein equivalent (attached by reaction of G3(4B) with fluorescein isothiocyanate, FITC) via thiourea bond as FITC followed by exhaustive glucoheptoamidation to get G3(4B27gh1F)...
November 25, 2016: Bioorganic & Medicinal Chemistry
https://www.readbyqxmd.com/read/27919267/targeted-drug-delivery-of-near-ir-fluorescent-doxorubicin-conjugated-poly-ethylene-glycol-bisphosphonate-nanoparticles-for-diagnosis-and-therapy-of-primary-and-metastatic-bone-cancer-in-a-mouse-model
#10
S Rudnick-Glick, E Corem-Salkmon, I Grinberg, S Margel
BACKGROUND: Most primary and metastatic bone tumors demonstrate increased osteoclast activity and bone resorption. Current treatment is based on a combination of surgery, radiotherapy and chemotherapy. Severe side effects are associated with chemotherapy due to use of high dosage and nonspecific uptake. Bisphosphonates have a strong affinity to Ca(2+) ions and are widely used in the treatment of bone disorders. RESULTS: We have engineered a unique biodegradable bisphosphonate nanoparticle (NPs) bearing two functional surface groups: (1) primary amine groups for covalent attachment of a dye/drug (e...
December 5, 2016: Journal of Nanobiotechnology
https://www.readbyqxmd.com/read/27915002/label-free-histamine-detection-with-nanofluidic-diodes-through-metal-ion-displacement-mechanism
#11
Mubarak Ali, Patricio Ramirez, Ivana Duznovic, Saima Nasir, Salvador Mafe, Wolfgang Ensinger
We design and characterize a nanofluidic device for the label-free specific detection of histamine neurotransmitter based on a metal ion displacement mechanism. The sensor consists of an asymmetric polymer nanopore fabricated via ion track-etching technique. The nanopore sensor surface having metal-nitrilotriacetic (NTA-Ni(2+)) chelates is obtained by covalent coupling of native carboxylic acid groups with Nα,Nα-bis(carboxymethyl)-l-lysine (BCML), followed by exposure to Ni(2+) ion solution. The BCML immobilization and subsequent Ni(2+) ion complexation with NTA moieties change the surface charge concentration, which has a significant impact on the current-voltage (I-V) curve after chemical modification of the nanopore...
November 28, 2016: Colloids and Surfaces. B, Biointerfaces
https://www.readbyqxmd.com/read/27914083/antibody-guided-molecular-imaging-of-infective-endocarditis
#12
Kenneth L Pinkston, Peng Gao, Kavindra V Singh, Ali Azhdarinia, Barbara E Murray, Eva M Sevick-Muraca, Barrett R Harvey
In this protocol, we describe the application of using a high affinity monoclonal antibody generated against the major pilin protein component of the pilin structure of Enterococcus faecalis as a PET imaging agent for enterococcal endocarditis detection. The anti-pilin -mAb 64Cu conjugate was able to specifically label enterococcal endocarditis vegetation in vivo in a rodent endocarditis model. By targeting pili, a covalently linked surface antigen extending from the bacterial surface, we provided evidence that gram-positive pilin represent a logical surface antigen to define or target an infectious agent for molecularly guided imaging...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/27896865/sensing-at-the-surface-of-graphene-field-effect-transistors
#13
Wangyang Fu, Lin Jiang, Erik P van Geest, Lia M C Lima, Grégory F Schneider
Recent research trends now offer new opportunities for developing the next generations of label-free biochemical sensors using graphene and other two-dimensional materials. While the physics of graphene transistors operated in electrolyte is well grounded, important chemical challenges still remain to be addressed, namely the impact of the chemical functionalizations of graphene on the key electrical parameters and the sensing performances. In fact, graphene - at least ideal graphene - is highly chemically inert...
November 29, 2016: Advanced Materials
https://www.readbyqxmd.com/read/27896766/multiplex-immunoassay-profiling
#14
Laurie Stephen
Multiplex immunoassays allow for the rapid profiling of biomarker proteins in biological fluids, using less sample and labor than single immunoassays. This chapter details the methods to develop and manufacture multiplex assays for the Luminex(®) platform. Although assay development is not included here, the same methods can be used to covalently couple antibodies to the Luminex beads and to label antibodies for the screening of sandwich pairs, if needed. The assay optimization, detection of cross-reactivity, and minimizing antibody interactions and matrix interferences will be addressed...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/27888685/label-free-piezoelectric-biosensor-for-prognosis-and-diagnosis-of-systemic-lupus-erythematosus
#15
Noelle M do Nascimento, Augusto Juste-Dolz, Elena Grau-García, Jose A Román-Ivorra, Rosa Puchades, Angel Maquieira, Sergi Morais, David Gimenez-Romero
An autoantigen piezoelectric sensor to quantify specific circulating autoantibodies in human serum is developed. The sensor consisted on a quartz crystal microbalance with dissipation monitoring (QCM-D) where TRIM21 and TROVE2 autoantigens were covalently immobilized, allowing the selective determination of autoantibodies for diagnosis and prognosis of Systemic Lupus Erythematosus (SLE). The sensitivity of the biosensor, measured as IC50 value, was 1.51U/mL and 0.32U/mL, for anti-TRIM21 and anti-TROVE2 circulating autoantibodies, respectively...
November 9, 2016: Biosensors & Bioelectronics
https://www.readbyqxmd.com/read/27886318/site-specific-covalent-capture-of-human-o-6-alkylguanine-dna-alkyltransferase-using-single-stranded-intrastrand-cross-linked-dna
#16
D K O'Flaherty, C J Wilds
A methodology is reported to conjugate human O(6)-alkylguanine-DNA-alkyltransferase (hAGT) to the 3'-end of DNA in excellent yields with short reaction times by using intrastrand cross-linked (IaCL) DNA probes. This strategy exploited the substrate specificity of hAGT to generate the desired DNA-protein covalent complex. IaCL DNA linking two thymidine residues, or linking a thymidine residue to a 2'-deoxyguanosine residue (either in a 5'→3' or 3'→5' fashion), lacking a phosphodiester linkage at the cross-linked site, were prepared using a phosphoramidite strategy followed by solid-phase synthesis...
November 25, 2016: Organic & Biomolecular Chemistry
https://www.readbyqxmd.com/read/27883258/bivalent-display-of-dicysteine-on-pna-for-homogenous-dna-rna-detection-via-in-situ-fluorescence-labelling
#17
Gemin Fang, Oliver Seitz
Fluorogenic probes that signal the presence of specific DNA or RNA sequences are key enabling tools for molecular disease diagnosis and imaging studies. Usually, at least one fluorophore is attached via covalent bonds to an oligonucleotide probe. However, the additional conjugation step increases costs. Herein we introduce a method that avoids the requirement for the preparation of fluorescence labeled oligonucleotides and provides the opportunity to alter the fluorogenic reporter dye without resynthesis. The method is based on adjacent hybridization of two dicysteine-containing peptide nucleic acid probes to form a bipartite tetracysteine motif which binds profluorescent bisarsenical dyes such as FIAsH, ReAsH or CrAsH...
November 24, 2016: Chembiochem: a European Journal of Chemical Biology
https://www.readbyqxmd.com/read/27879639/disposable-amperometric-immunosensor-for-the-determination-of-human-p53-protein-in-cell-lysates-using-magnetic-micro-carriers
#18
María Pedrero, F Javier Manuel de Villena, Cristina Muñoz-San Martín, Susana Campuzano, María Garranzo-Asensio, Rodrigo Barderas, José M Pingarrón
An amperometric magnetoimmunosensor for the determination of human p53 protein is described in this work using a sandwich configuration involving the covalent immobilization of a specific capture antibody onto activated carboxylic-modified magnetic beads (HOOC-MBs) and incubation of the modified MBs with a mixture of the target protein and horseradish peroxidase-labeled antibody (HRP-anti-p53). The resulting modified MBs are captured by a magnet placed under the surface of a disposable carbon screen-printed electrode (SPCE) and the amperometric responses are measured at -0...
November 21, 2016: Biosensors
https://www.readbyqxmd.com/read/27873206/discovery-of-rna-binding-small-molecules-using-small-molecule-microarrays
#19
Colleen M Connelly, Fardokht A Abulwerdi, John S Schneekloth
New methods to identify RNA-binding small molecules open yet unexplored opportunities for the pharmacological modulation of RNA-driven biology and disease states. One such approach is the use of small molecule microarrays (SMMs). Typically, SMMs are generated by spatially arraying and covalently linking a library of small molecules to a glass surface. Next, incubation of the arrays with a fluorescently labeled RNA reveals binding interactions that are detected upon slide imaging. The relative ease with which SMMs are manufactured enables the screening of multiple oligonucleotides in parallel against tens of thousands of small molecules, providing information about both binding and selectivity of identified RNA-small molecule interactions...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/27872191/structural-and-biochemical-analyses-reveal-the-mechanism-of-glutathione-s-transferase-pi-1-inhibition-by-the-anti-cancer-compound-piperlongumine
#20
Wayne Harshbarger, Sudershan Gondi, Scott B Ficarro, John Hunter, Durga Udayakumar, Deepak Gurbani, William Singer, Yan Liu, Lianbo Li, Jarrod A Marto, Kenneth D Westover
Glutathione S-transferase pi 1 (GSTP1), is frequently overexpressed in cancerous tumors and is a putative target of the plant compound piperlongumine (PL), which contains two reactive olefins and inhibits proliferation in cancer cells but not normal cells. PL exposure of cancer cells results in increased reactive oxygen species and decreased glutathione (GSH). This data in tandem with other information led to the conclusion that PL inhibits GSTP1, which forms covalent bonds between GSH and various electrophilic compounds, through covalent adduct formation at PLs C7-C8 olefin, while PLs C2-C3 olefin was postulated to react with GSH...
November 21, 2016: Journal of Biological Chemistry
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