Name: hplc measurement of the dna oxidation biomarker, 8-oxo-7,8-dihydro-2’-deoxyguanosine, in cultured cells and animal tissues
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Description: Watch this Scientific Journal Video about HPLC Measurement of the DNA Oxidation Biomarker, 8-oxo-7,8-dihydro-2Ã¢â‚¬â„¢-deoxyguanosine, in Cultured Cells and Animal Tissues at JoVE.com

Esta investigación fue financiada por la Iniciativa de Salud de Canadá Genómica Investigación y Desarrollo (GRDI) y la Estrategia de Regulación Canadiense de Biotecnología (letras CRSB). Los autores no tienen ningún conflicto de intereses.

<ol>
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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+an+HPLC+method+with+electrochemical+detection+of+femtomoles+of+8-oxo-7,8-dihydroguanine+and+8-oxo-7,8-dihydro-2'-deoxyguanosine+in+the+presence+of+uric+acid.">Development of an HPLC method with electrochemical detection of femtomoles of 8-oxo-7,8-dihydroguanine and 8-oxo-7,8-dihydro-2'-deoxyguanosine in the presence of uric acid.</a> Talanta. 63 (2), 323-331 (2004).</li><li>Ravanat, J. -. L., Turesky, R. J., Gremaud, E., Trudel, L. J., Stadler, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+8-oxoguanine+in+DNA+by+gas+chromatography-mass+spectrometry+and+HPLC-electrochemical+detection:+overestimation+of+the+background+level+of+the+oxidized+base+by+the+gas+chromatography-mass+spectrometry+assay.">Determination of 8-oxoguanine in DNA by gas chromatography-mass spectrometry and HPLC-electrochemical detection: overestimation of the background level of the oxidized base by the gas chromatography-mass spectrometry assay.</a> Chem. Res. Toxicol. 8 (8), 1039-1045 (1995).</li><li>Kawanishi, S., Murata, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+DNA+damage+induced+by+bromate+differs+from+general+types+of+oxidative+stress.">Mechanism of DNA damage induced by bromate differs from general types of oxidative stress.</a> Toxicology. 221 (2-3), 172-178 (2006).</li><li>Tahara, S., Kaneko, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Susceptibility+of+mouse+splenic+cells+to+oxidative+DNA+damage+by+x-ray+irradiation.">Susceptibility of mouse splenic cells to oxidative DNA damage by x-ray irradiation.</a> Biol. Pharm. Bull. 27 (1), 105-108 (2004).</li><li>Garratt, L. W., Mistry, V., Singh, R., Sandhu, J. K., Sheil, B., Cooke, M. S., Sly, P. 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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+oxidatively+generated+base+damage+in+cellular+DNA.">Measurement of oxidatively generated base damage in cellular DNA.</a> Mutat Res. 711 (1-2), 3-12 (2011).</li><li>Chomczynski, P., Mackey, K., Drews, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNAzol:+a+reagent+for+the+rapid+isolation+of+genomic+DNA.">DNAzol: a reagent for the rapid isolation of genomic DNA.</a> Biotechniques. 22 (3), 550-553 (1997).</li><li>Collins, A. R., Cadet, J., M&#246;ller, L., Poulsen, H. 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No conflict of interest or competing financial interests declared.

Aunque 8-oxo-dGuo ha sido reportado como un biomarcador útil de la oxidación del ADN, su cuantificación fiable puede suponer un reto. Aunque existen varios métodos publicados, hay una necesidad de una visión global, descriptivo de protocolo para permitir a los investigadores a implementar el método en sus laboratorios. Aquí presentamos un resumen detallado de un protocolo basado en HPLC que permita a los nuevos usuarios para establecer un método eficaz para la detección y cuantificación de 8-oxo-dGuo. <p c...

Especies reactivas del oxígeno (ROS), cuyos niveles de estado estacionario puede aumentar durante muchas condiciones patológicas y el metabolismo xenotoxic, contribuyen a una mayor frecuencia de daño oxidativo del ADN. Entre varios productos de oxidación posibles nucleobases, daño oxidativo del ADN fácilmente se puede medir usando el marcador estable 8-oxo-7,8-dihidro-2 &#39;desoxiguanosina (8-oxo-dGuo), que es una de las formas oxidadas de 2&#39; -deoxyguanosine (dGuo) 1. 8-oxo-dGuo es el más abundante lesión del ADN 2 y, por lo tanto, ha sido estudiado para mayor detalle como un biomarcador oxidación del ADN a pesar de la existencia de productos de oxidación de ADN múltiple 3. En los seres humanos, este daño puede ser reparado a través de la reparación por escisión de base por 8-oxoguanina glicosilasa 1 (hOGG1) 4. Si se deja sin reparar, 8-oxo-dGuo puede contribuir a la formación de mutaciones de sustitución de pares de bases (es decir, G a T transversiones) 4. Es importante destacar que, 8-oxo-dGuo es un marcador establecido fodaño en el ADN r en relación con la iniciación y promoción de la carcinogénesis 2. Por lo tanto, la cuantificación precisa de 8-oxo-dGuo es un biomarcador útil y deseable de daño oxidativo del ADN 5. Hay una confusión generalizada en la literatura con respecto a los nombres correctos de las formas dañadas oxidativamente de 2-desoxiguanosina y, por otra parte, el nombre correcto del compuesto (s) mide rutinariamente como un biomarcador de daño oxidativo del ADN 6. Los 6,8-diceto-enol y 6, 8-ceto formas tautoméricas de 8-oxo-dGuo (mostrado en la Figura 1) son los dos tautómeros más prominentes discutidos en la literatura 5,7. La forma de 6,8-diceto es la forma más prominente a pH fisiológico de 7,4, y es el producto de oxidación de ADN más prominente 7. Por lo tanto, 8-oxo-dGuo, en lugar de 8-hidroxi-dGuo es el nombre más apropiado para este producto de oxidación 6. También es importante señalar que 2-desoxiguanosina (dGuo), en lugar de nucleobase guanina (Gua) o guanosina ribonucleósido (Guo), respectivamente, se detecta por la mayoría de los métodos de 6. Detección y cuantificación de 8-oxo-dGuo exacta es difícil debido a: i) la variabilidad en la digestión de la muestra de ADN, ii) la oxidación adventicia de dGuo a 8-oxo-dGuo que puede ocurrir durante la preparación de la muestra, y iii) la necesidad para la validación efectiva del método HPLC-ED analítica 8. En este protocolo, que tuvo como objetivo a lograr i) proporcionando condiciones favorables para la digestión completa del ADN y ii) por la inclusión quelante de metal y soluciones quelantes tratados y un reactivo DNA-aislamiento especial, mientras iii) fue sólo parcialmente abordados por la inclusión de controles positivos y, por tanto proporcionando que el método es capaz de detectar 8-oxo-dGuo en muestras biológicas. Además de validación está más allá del alcance de este documento. Sin embargo, estamos seguros de que este protocolo ayudará a la prospectivalos usuarios a determinar el grado en que ellos necesitan para validar formalmente el protocolo, dependiendo de sus fines. Una lista de los pasos requeridos para la validación formal del método está provisto además. Durante el desarrollo e implementación de un método para la detección de 8-oxo-dGuo, métodos publicados fueron revisados ​​y consolidados. Por lo tanto, este método elimina la necesidad de reunir información de varias fuentes publicadas que a menudo carecen de importantes detalles experimentales mientras que también proporciona medios rápidos y directos de las pruebas si el método para la detección y cuantificación de 8-oxo-dGuo ha sido adoptado con éxito. Este método adaptado fue empleado con éxito para analizar muestras de ADN de las células cultivadas y el tejido murino. Este artículo de vídeo ayudará a otros grupos en el establecimiento de un método eficaz para la detección fiable y cuantificación de 8-oxo-dGuo por HPLC-ED.

<table><tbody><tr><td>8-oxo-dGuo standard</td><td>Cayman Chemical Company</td><td>89320</td><td>Inappropriately referred to as &quot;8-hydroxy-2&#039;-deoxy Guanosine&quot;; see &lt;strong&gt;Fig. 1&lt;/strong&gt; and text for details.</td></tr><tr><td>Alkaline phosphatase</td><td>Sigma-Aldrich</td><td>P5931</td><td>From &lt;em&gt;E. coli&lt;/em&gt;</td></tr><tr><td>Chelex 100</td><td>Sigma-Aldrich</td><td>C7901</td><td>Chelates heavy metals</td></tr><tr><td>Desferoxamine mesylate</td><td>Sigma-Aldrich</td><td>D9533</td><td></td></tr><tr><td>dGuo standard</td><td>Sigma-Aldrich</td><td>D7145</td><td></td></tr><tr><td>Dibasic sodium phosphate</td><td>Sigma-Aldrich</td><td>S9390</td><td></td></tr><tr><td>DNA from salmon sperm</td><td>Sigma-Aldrich</td><td>D1626</td><td>Sodium salt</td></tr><tr><td>DNase I</td><td>Sigma-Aldrich</td><td>D4527</td><td>TypeII, from bovine pancreas</td></tr><tr><td>DNAzol</td><td>Invitrogen</td><td>10503-27</td><td></td></tr><tr><td>Ethylenediaminetetraacetic acid disodium salt dihydrate (EDTA)</td><td>Sigma-Aldrich</td><td>E4884</td><td>The compound would not completely dissolve until solution pH is adjusted to 8.0 (&lt;em&gt;e.g.&lt;/em&gt; with NaOH)</td></tr><tr><td>F12-K media</td><td>ATCC</td><td>30-2004</td><td></td></tr><tr><td>Foetal bovine serum</td><td>ATCC</td><td>30-2020</td><td></td></tr><tr><td>Guard column</td><td>Chromatographic Specialties</td><td>YBA 99S03 0204GC</td><td>Protects colum from contamination; may also lead to pressure build-up</td></tr><tr><td>Magnesium chloride</td><td>Sigma-Aldrich</td><td>M8266</td><td></td></tr><tr><td>Monobasic sodium phosphate</td><td>Sigma-Aldrich</td><td>S9638</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Invitrogen</td><td>15140-122</td><td></td></tr><tr><td>Phosphate buffered saline</td><td>Invitrogen</td><td>15190-250</td><td></td></tr><tr><td>Phosphodiesterase I enzyme&amp;nbsp;</td><td>Sigma-Aldrich</td><td>P3243</td><td>Type II from Crotalus adamaneus venom</td></tr><tr><td>Teflon homogenizer</td><td>Thomas Scientific</td><td>7724T-1 or 7724T-5 for 1 or 5 mL, respectively</td><td>Volume (holding capacity) depends on the amount of sample to be processed.</td></tr><tr><td>Trypsin</td><td>Invitrogen</td><td>15050-065</td><td></td></tr><tr><td>YMC-BASIC column with bonded spherical silica</td><td>Chromatographic Specialties</td><td>YBA 99S03 1546WT</td><td></td></tr></tbody></table>

hplc measurement of the dna oxidation biomarker, 8-oxo-7,8-dihydro-2&#8217;-deoxyguanosine, in cultured cells and animal tissues

El estrés oxidativo está asociado con muchos procesos fisiológicos y patológicos, así como el metabolismo de xenobióticos, que conduce a la oxidación de biomacromoléculas, incluyendo ADN. Por lo tanto, la detección eficiente de la oxidación del ADN es importante para una variedad de disciplinas de investigación, incluyendo la medicina y toxicología. Un biomarcador común de ADN oxidativamente dañado es 8-oxo-7,8-dihidro-2 &#39;desoxiguanosina (8-oxo-dGuo; a menudo denominado erróneamente como 8-hidroxi-2&#39; desoxiguanosina (8-OH-dGuo o 8 oxo-DG)). Se han descrito varios protocolos para la medición de 8-oxo-dGuo por cromatografía líquida de alta presión con detección electroquímica (HPLC-ED). Sin embargo, éstos se aplicaron principalmente a ADN purificado tratado con pro-oxidantes. Además, debido a las diferencias metodológicas entre los laboratorios, debido principalmente a las diferencias en equipo analítico, la adopción de métodos publicados para la detección de 8-oxo-dGuo por HPLC-ED requiere la optimización cuidadosa de cada laboratorio. LAprotocolo integral, que describe un procedimiento de este tipo de optimización, es insuficiente. Aquí, un protocolo detallado se describe para la detección de 8-oxo-dGuo por HPLC-ED, en el ADN de células cultivadas o tejidos animales. Se ilustra cómo la preparación de muestras de ADN se puede optimizar fácilmente y rápidamente para minimizar la oxidación indeseable de ADN que puede ocurrir durante la preparación de la muestra. Este protocolo muestra cómo detectar 8-oxo-dGuo en células cultivadas humanos adenocarcinoma alveolar (es decir, células A549) tratados con el agente oxidante KBrO 3, y del bazo de ratones expuestos a la dibenzo hidrocarburo aromático policíclico (def, p) criseno (DBC, antes conocido como dibenzo (a, l) pireno, DALP). En general, este trabajo ilustra cómo una metodología HPLC-ED se puede optimizar fácilmente para la detección de 8-oxo-dGuo en muestras biológicas.

dGuo fue observado para tener un tiempo de retención de 4,7 min mientras que 8-oxo-dGuo tenía un tiempo de retención de aproximadamente 6,4 min (Figura 2A y B). Hay una diferencia sobre 1.000 veces en las alturas de los picos entre los dos analitos, como se ve en la Figura 2C. Voltamogramas para 8-oxo-dGuo y dGuo se obtuvieron por las normas de funcionamiento a un potencial de trabajo en el intervalo de 0,2 a 1,1 V. El potencial de trabajo óptimo para 8-oxo-dGuo se ...

Watch this Scientific Journal Video about HPLC Measurement of the DNA Oxidation Biomarker, 8-oxo-7,8-dihydro-2Ã¢â‚¬â„¢-deoxyguanosine, in Cultured Cells and Animal Tissues at JoVE.com

HPLC Measurement of the DNA Oxidation Biomarker, 8-oxo-7,8-dihydro-2&#8217;-deoxyguanosine, in Cultured Cells and Animal Tissues

El objetivo de este protocolo es la detección del marcador de la oxidación del ADN, 8-oxo-7,8-dihidro-2 &#39;desoxiguanosina (8-oxo-dGuo) por HPLC-ED, en el ADN de células cultivadas o tejidos animales.

HPLC Medición de la oxidación del ADN de biomarcadores, 8-oxo-7,8-dihidro-2 &#39;desoxiguanosina, en células cultivadas y tejidos animales

Oxidative stress is associated with many physiological and pathological processes, as well as xenobiotic metabolism, leading to the oxidation of ...

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Chemistry

26.4K vistas.  Health Canada.  El objetivo de este protocolo es la detección del marcador de la oxidación del ADN, 8-oxo-7,8-dihidro-2 'desoxiguanosina (8-oxo-dGuo) por HPLC-ED, en el ADN de células cultivadas o tejidos animales. 

Video: HPLC Measurement of the DNA Oxidation Biomarker, 8-oxo-7,8-dihydro-2’-deoxyguanosine, in Cultured Cells and Animal Tissues

Free Radicals in Chemical Biology: from Chemical Behavior to Biomarker Development

The involvement of free radicals in life sciences has constantly increased with time and has been connected to several physiological and pathological processes. This subject embraces diverse scientific areas, spanning from physical, biological and bioorganic chemistry to biology and medicine, with applications to the amelioration of quality of life, health and aging. Multidisciplinary skills are required for the full investigation of the many facets of radical processes in the biological environment and chemical knowledge plays a crucial role in unveiling basic processes and mechanisms. We developed a chemical biology approach able to connect free radical chemical reactivity with biological processes, providing information on the mechanistic pathways and products. The core of this approach is the design of biomimetic models to study biomolecule behavior (lipids, nucleic acids and proteins) in aqueous systems, obtaining insights of the reaction pathways as well as building up molecular libraries of the free radical reaction products. This context can be successfully used for biomarker discovery and examples are provided with two classes of compounds: mono-trans isomers of cholesteryl esters, which are synthesized and used as references for detection in human plasma, and purine 5',8-cyclo-2'-deoxyribonucleosides, prepared and used as reference in the protocol for detection of such lesions in DNA samples, after ionizing radiations or obtained from different health conditions.

Radical-based biomimetic chemistry has been applied to building-up libraries necessary for biomarker development.

Radicales Libres en Biología Química: del comportamiento químico al desarrollo de biomarcadores

The involvement of free radicals in life sciences has constantly increased with time and has been connected to several physiological and pathological ...

Investigación

Química

Steady-state, Pre-steady-state, and Single-turnover Kinetic Measurement for DNA Glycosylase Activity

Human 8-oxoguanine DNA glycosylase (OGG1) excises the mutagenic oxidative DNA lesion 8-oxo-7,8-dihydroguanine (8-oxoG) from DNA. Kinetic characterization of OGG1 is undertaken to measure the rates of 8-oxoG excision and product release. When the OGG1 concentration is lower than substrate DNA, time courses of product formation are biphasic; a rapid exponential phase (i.e. burst) of product formation is followed by a linear steady-state phase. The initial burst of product formation corresponds to the concentration of enzyme properly engaged on the substrate, and the burst amplitude depends on the concentration of enzyme. The first-order rate constant of the burst corresponds to the intrinsic rate of 8-oxoG excision and the slower steady-state rate measures the rate of product release (product DNA dissociation rate constant, koff). Here, we describe steady-state, pre-steady-state, and single-turnover approaches to isolate and measure specific steps during OGG1 catalytic cycling. A fluorescent labeled lesion-containing oligonucleotide and purified OGG1 are used to facilitate precise kinetic measurements. Since low enzyme concentrations are used to make steady-state measurements, manual mixing of reagents and quenching of the reaction can be performed to ascertain the steady-state rate (koff). Additionally, extrapolation of the steady-state rate to a point on the ordinate at zero time indicates that a burst of product formation occurred during the first turnover (i.e. y-intercept is positive). The first-order rate constant of the exponential burst phase can be measured using a rapid mixing and quenching technique that examines the amount of product formed at short time intervals (&lt;1 sec) before the steady-state phase and corresponds to the rate of 8-oxoG excision (i.e. chemistry). The chemical step can also be measured using a single-turnover approach where catalytic cycling is prevented by saturating substrate DNA with enzyme (E&gt;S). These approaches can measure elementary rate constants that influence the efficiency of removal of a DNA lesion.

Time courses for the glycosylase activity of 8-oxoguanine DNA glycosylase are biphasic exhibiting a burst of product formation and a linear steady-state phase. Utilizing quench-flow techniques, the burst and the steady-state rates can be measured, which correspond to excision of 8-oxoguanine and release of the glycosylase from the product DNA, respectively.

Medición cinética de estado estable, Pre-estado de equilibrio, y un solo volumen de negocios de ADN glicosilasa Actividad

Human 8-oxoguanine DNA glycosylase (OGG1) excises the mutagenic oxidative DNA lesion 8-oxo-7,8-dihydroguanine (8-oxoG) from DNA. Kinetic characterization ...

Folding and Characterization of a Bio-responsive Robot from DNA Origami

The DNA nanorobot is a hollow hexagonal nanometric device, designed to open in response to specific stimuli and present cargo sequestered inside. Both stimuli and cargo can be tailored according to specific needs. Here we describe the DNA nanorobot fabrication protocol, with the use of the DNA origami technique. The procedure initiates by mixing short single-strand DNA staples into a stock mixture which is then added to a long, circular, single-strand DNA scaffold in presence of a folding buffer. A standard thermo cycler is programmed to gradually lower the mixing reaction temperature to facilitate the staples-to-scaffold annealing, which is the guiding force behind the folding of the nanorobot. Once the 60 hr folding reaction is complete, excess staples are discarded using a centrifugal filter, followed by visualization via agarose-gel electrophoresis (AGE). Finally, successful fabrication of the nanorobot is verified by transmission electron microscopy (TEM), with the use of uranyl-formate as negative stain.

DNA origami is a powerful method for fabricating precise nanoscale objects by programming the self-assembly of DNA molecules. Here we describe a protocol for the folding of a bio-responsive robot from DNA origami, its purification and negative staining for transmission electron microscopic imaging (TEM).

Plegable y caracterización de un Robot Bio-sensible de ADN Origami

The DNA nanorobot is a hollow hexagonal nanometric device, designed to open in response to specific stimuli and present cargo sequestered inside. Both ...

HPLC-based Assay to Monitor Extracellular Nucleotide/Nucleoside Metabolism in Human Chronic Lymphocytic Leukemia Cells

This method describes a sensitive, specific, reliable and reproducible reverse phase high-performance liquid chromatography (RP-HPLC) assay developed and validated for the quantification of extracellular purine nucleotides and nucleosides produced by purified chronic lymphocytic leukemia (CLL) cells under different culture conditions. The chromatographic separation of adenosine 5&#39;-monophosphate (AMP), adenosine (ADO) and inosine (INO) is performed at RT on a silica-based, reversed-phase column that is used for polar compound retention. The method includes a binary mobile phase, which consists of 7 mM ammonium acetate and acetonitrile with a flow rate of 1.00 ml/min. The eluates are monitored using a Photodiode Array UV detector set at 260 nm. A standard calibration curve is generated to calculate the equation for the analytical quantification of each purine compound. System control, data acquisition and analysis are then performed. Applying this protocol, AMP, INO and ADO elute at 7, 11 and 11.9 min, respectively, and the total run time for each sample is 20 min. This protocol may be applied to different cell types and cell lines (both suspension and adherent), using culture media as matrix. The advantages are easy and fast sample preparation and the requirement of a small amount of supernatant for analysis. Furthermore, the use of a serum-free medium allows skipping the protein precipitation step with acetonitrile that impacts the final concentration of purine compounds. One of the limitations of the method is the requirement of the equilibration column run before each single sample run, making the total run time of the experiment longer and preventing high throughput screening applications.

The protocol described here represents an easy and reproducible method that employs reverse phase high-performance liquid chromatography (RP-HPLC) to measure purine metabolism on chronic lymphocytic leukemia (CLL) cells cultured under different conditions.

Ensayo basado en HPLC para supervisar extracelular de nucleótidos / nucleósidos de metabolismo en las células de la leucemia crónica humana linfocítica

This method describes a sensitive, specific, reliable and reproducible reverse phase high-performance liquid chromatography (RP-HPLC) assay developed and ...

Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry

The chemical linking or bioconjugation of proteins to fluorescent dyes, drugs, polymers and other proteins has a broad range of applications, such as the development of antibody drug conjugates (ADCs) and nanomedicine, fluorescent microscopy and systems chemistry. For many of these applications, specificity of the bioconjugation method used is of prime concern. The Michael addition of maleimides with cysteine(s) on the target proteins is highly selective and proceeds rapidly under mild conditions, making it one of the most popular methods for protein bioconjugation.
We demonstrate here the modification of the only surface-accessible cysteine residue on yeast cytochrome c with a ruthenium(II) bisterpyridine maleimide. The protein bioconjugation is verified by gel electrophoresis and purified by aqueous-based fast protein liquid chromatography in 27% yield of isolated protein material. Structural characterization with MALDI-TOF MS and UV-Vis is then used to verify that the bioconjugation is successful. The protocol shown here is easily applicable to other cysteine - maleimide coupling of proteins to other proteins, dyes, drugs or polymers.

Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry

This protocol details the important steps required for the bioconjugation of a cysteine containing protein to a maleimide, including reagent purification, reaction conditions, bioconjugate purification and bioconjugate characterization.

Síntesis de Proteínas bioconjugados<em&gt; vía</em&gt; Química cisteína-maleimida

The chemical linking or bioconjugation of proteins to fluorescent dyes, drugs, polymers and other proteins has a broad range of applications, such as the ...

Determining the Chemical Composition of Corrosion Inhibitor/Metal Interfaces with XPS: Minimizing Post Immersion Oxidation

An approach for acquiring more reliable X-ray photoelectron spectroscopy data from corrosion inhibitor/metal interfaces is described. More specifically, the focus is on metallic substrates immersed in acidic solutions containing organic corrosion inhibitors, as these systems can be particularly sensitive to oxidation following removal from solution. To minimize the likelihood of such degradation, samples are removed from solution within a glove box purged with inert gas, either N2 or Ar. The glove box is directly attached to the load-lock of the ultra-high vacuum X-ray photoelectron spectroscopy instrument, avoiding any exposure to the ambient laboratory atmosphere, and thus reducing the possibility of post immersion substrate oxidation. On this basis, one can be more certain that the X-ray photoelectron spectroscopy features observed are likely to be representative of the in situ submerged scenario, e.g. the oxidation state of the metal is not modified.

A protocol to avoid the oxidation of metallic substrates during sample transfer from an inhibited acidic solution to an X-ray photoelectron spectrometer is presented.

La determinación de la composición química del inhibidor de corrosión / Metal Interfaces con XPS: Reducción al mínimo del anuncio Inmersión Oxidación

An approach for acquiring more reliable X-ray photoelectron spectroscopy data from corrosion inhibitor/metal interfaces is described. More specifically, ...

Fabrication and Testing of Photonic Thermometers

In recent years, a push for developing novel silicon photonic devices for telecommunications has generated a vast knowledge base that is now being leveraged for developing sophisticated photonic sensors. Silicon photonic sensors seek to exploit the strong confinement of light in nano-waveguides to transduce changes in physical state to changes in resonance frequency. In the case of thermometry, the thermo-optic coefficient, i.e., changes in refractive index due to temperature, causes the resonant frequency of the photonic device such as a Bragg grating to drift with temperature. We are developing a suite of photonic devices that leverage recent advances in telecom compatible light sources to fabricate cost-effective photonic temperature sensors, which can be deployed in a wide variety of settings ranging from controlled laboratory conditions, to the noisy environment of a factory floor or a residence. In this manuscript, we detail our protocol for the fabrication and testing of photonic thermometers.

We describe the process of fabrication and testing of photonic thermometers.

Fabricación y pruebas de termómetros fotónicos

In recent years, a push for developing novel silicon photonic devices for telecommunications has generated a vast knowledge base that is now being ...

Simultaneous Measurement of HDAC1 and HDAC6 Activity in HeLa Cells Using UHPLC-MS

The search for new histone deacetylase (HDAC) inhibitors is of increasing interest in drug discovery. Isoform selectivity has been in the spotlight since the approval of romidepsin, a class I HDAC inhibitor for cancer therapy, and the clinical investigation of HDAC6-specific inhibitors for multiple myeloma. The present method is used to determine the inhibitory activity of test compounds on HDAC1 and HDAC6 in cells. The isoform activity is measured using the ultra-high-performance liquid chromatography &#8211; mass spectrometry (UHPLC-MS) analysis of specific substrates incubated with treated and untreated HeLa cells. The method has the advantage of reflecting the endogenous HDAC activity within the cell environment, in contrast to cell-free biochemical assays conducted on isolated isoforms. Moreover, because it is based on the quantification of synthetic substrates, the method does not require the antibody recognition of endogenous acetylated proteins. It is easily adaptable to several cell lines and an automated process. The method has already proved useful in finding HDAC6-selective compounds in neuroblasts. Representative results are shown here with the standard HDAC inhibitors trichostatin A (non-specific), MS275 (HDAC1-specific), and tubastatin A (HDAC6-specific) using HeLa cells.

The present method serves to identify isoform-specific inhibitors of histone deacetylases (HDAC) in HeLa cells by the UHPLC-MS analysis of multiple substrates. This is an antibody-free method developed to reflect HDAC1 and HDAC6 activity in the living cell environment, in contrast to single-isoform cell-free assays.

Medición simultánea de HDAC1 y HDAC6 actividad en HeLa células usando UHPLC-MS

The search for new histone deacetylase (HDAC) inhibitors is of increasing interest in drug discovery. Isoform selectivity has been in the spotlight since ...

Determinación del grado de daño oxidativo del ADN en células MCF-7

Quantifying the Level of 8-oxo-dG Using ELISA Assay to Evaluate Oxidative DNA Damage in MCF-7 Cells

8-Oxo-7,8-dihydro-2&#39;-deoxyguanosine (8-oxo-dG) base is the predominant form of commonly observed DNA oxidative damage. DNA impairment profoundly impacts gene expression and serves as a pivotal factor in stimulating neurodegenerative disorders, cancer, and aging. Therefore, precise quantification of 8-oxoG has clinical significance in the investigation of DNA damage detection methodologies. However, at present, the existing approaches for 8-oxoG detection pose challenges in terms of convenience, expediency, affordability, and heightened sensitivity. We employed the sandwich enzyme-linked immunosorbent assay (ELISA) technique, a highly efficient and swift colorimetric method, to detect variations in 8-oxo-dG content in MCF-7 cell samples stimulated with different concentrations of hydrogen peroxide (H2O2). We determined the concentration of H2O2 that induced oxidative damage in MCF-7 cells by detecting its IC50 value in MCF-7 cells. Subsequently, we treated MCF-7 cells with 0, 0.25, and 0.75 mM H2O2 for 12 h and extracted 8-oxo-dG from the cells. Finally, the samples were subjected to ELISA. Following a series of steps, including plate spreading, washing, incubation, color development, termination of the reaction, and data collection, we successfully detected changes in the 8-oxo-dG content in MCF-7 cells induced by H2O2. Through such endeavors, we aim to establish a method to evaluate the degree of DNA oxidative damage within cell samples and, in doing so, advance the development of more expedient and convenient approaches for DNA damage detection. This endeavor is poised to make a meaningful contribution to the exploration of associative analyses between DNA oxidative damage and various domains, including clinical research on diseases and the detection of toxic substances.

Autor destacado: Evaluación cuantitativa de 8-oxo-dG en células MCF-7 mediante ELISA

This protocol describes an efficient method for quantitatively detecting DNA oxidative damage in MCF-7 cells by ELISA technology.

Cuantificación del nivel de 8-oxo-dG mediante el ensayo ELISA para evaluar el daño oxidativo del ADN en células MCF-7

8-Oxo-7,8-dihydro-2&#39;-deoxyguanosine (8-oxo-dG) base is the predominant form of commonly observed DNA oxidative damage. DNA impairment profoundly ...

HPLC Medición de la oxidación del ADN de biomarcadores, 8-oxo-7,8-dihidro-2 'desoxiguanosina, en células cultivadas y tejidos animales

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Radicales Libres en Biología Química: del comportamiento químico al desarrollo de biomarcadores

Medición cinética de estado estable, Pre-estado de equilibrio, y un solo volumen de negocios de ADN glicosilasa Actividad

Plegable y caracterización de un Robot Bio-sensible de ADN Origami

Ensayo basado en HPLC para supervisar extracelular de nucleótidos / nucleósidos de metabolismo en las células de la leucemia crónica humana linfocítica

Síntesis de Proteínas bioconjugados

La determinación de la composición química del inhibidor de corrosión / Metal Interfaces con XPS: Reducción al mínimo del anuncio Inmersión Oxidación

Fabricación y pruebas de termómetros fotónicos

Medición simultánea de HDAC1 y HDAC6 actividad en HeLa células usando UHPLC-MS

Cuantificación del nivel de 8-oxo-dG mediante el ensayo ELISA para evaluar el daño oxidativo del ADN en células MCF-7

Cuantificación del nivel de 8-oxo-dG mediante el ensayo ELISA para evaluar el daño oxidativo del ADN en células MCF-7