מחקר זה מומן על ידי משרד הבריאות הקנדית ג&#39;נומיקס המחקר ופיתוח היוזמה (GRDI) ותקינת האסטרטגיה הקנדית לביוטכנולוגיה (CRSB). יש הסופרים אין ניגוד האינטרסים.

<ol>
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No conflict of interest or competing financial interests declared.

למרות 8-אוקסו-dGuo דווח כסמן ביולוגי שימושי של חמצון ה- DNA, הכימות אמין שלה יכול להוות אתגר. למרות כמה שיטות שפורסמו קיימות, יש צורך בסקירה מקיפה, תיאורים של פרוטוקול להתיר לחוקרים לפרוס את השיטה במעבדות שלהם. כאן אנו מציגים סקירה מפורטת של פרוטוקול מבוסס HPLC שיאפשר למשתמש...

מינים תגובתי חמצן (ROS), מצב יציב שיכולים להגדיל את הרמות בהרבה מצבים פתולוגיים וחילוף חומרי xenotoxic, תורמים לשכיחות מוגברת של נזק לדנ&quot;א חמצוני. בין כמה מוצרי חמצון nucleobases האפשרי, נזק לדנ&quot;א חמצוני יכול בקלות למדוד באמצעות הסמן היציב 8-אוקסו-7,8-Dihydro-2&#39;-deoxyguanosine (8-אוקסו-dGuo), שהוא אחת מצורות חמצון של 2 &#39; -deoxyguanosine (dGuo) 1. 8-אוקסו-dGuo הוא נגע DNA הנפוץ ביותר 2, ולכן, כבר למד לפירוט רב יותר כסמן ביולוגי חמצון ה- DNA למרות קיומם של מוצרי חמצון ה- DNA המרובים 3. בבני אדם, נזק זה ניתן לתיקון באמצעות תיקון כריתת בסיס על ידי glycosylase 8-oxoguanine 1 (hOGG1) 4. אם עזב מתוקן, 8-אוקסו-dGuo יכול לתרום להיווצרותן של מוטציות זוג-החלפת בסיס (כלומר, G לT transversions) 4. חשוב לציין, 8-אוקסו-dGuo הוא סמן הוקם FOנזק לדנ&quot;א r ביחס לייזום והקידום של היווצרות הסרטן 2. לכן, כימות מדויק של 8-אוקסו-dGuo הוא סמן ביולוגי שימושי ורצוי של נזק לדנ&quot;א חמצוני 5. יש בלבול נפוץ בספרות בנוגע לשמות הנכונים לצורות oxidatively פגועות של 2-deoxyguanosine ו, יתר על כן, את השם הנכון של המתחם (ים) שנמדדו באופן שגרתי כסמן ביולוגי של נזק לדנ&quot;א חמצוני 6. צורות tautomeric 8-קטו 6,8-diketo ו6-enol, של 8-אוקסו-dGuo (מוצג באיור 1) הן שני tautomers הבולט ביותר שנדון בספרות 5,7. טופס 6,8-diketo הוא הצורה הבולטת ביותר ב- pH הפיזיולוגי של 7.4, והוא מוצר חמצון ה- DNA הבולט 7. לכן, 8-אוקסו-dGuo, במקום 8-הידרוקסי-dGuo הוא השם המתאים ביותר למוצר חמצון זה 6. כמו כן, חשוב לשים לב ש2-deoxyguanosine (dGuo), ולא nucleobגואנין ASE (גואה) או guanosine ribonucleoside (גואו), בהתאמה, הוא זוהה על ידי רוב השיטות 6. איתור וכימות של 8-אוקסו-dGuo מדויקים הוא מאתגר עקב: השתנות i) בעיכול של דגימת DNA, ii) חמצון adventitious של dGuo 8-אוקסו-dGuo שיכול להתרחש במהלך הכנת מדגם, וiii) את הצורך לאימות יעילה של שיטת HPLC-ED אנליטיים 8. בפרוטוקול זה, אנו חותרים להשגת i) על ידי מתן תנאים, נוחים לעיכול DNA מלא וii) על ידי chelator הכללת המתכת ופתרונות שטופל chelator ומגיב בידוד-דנ&quot;א מיוחד, תוך iii) היה ממוען רק באופן חלקי על ידי הכללת בקרות חיוביות וכך לספק כי השיטה היא מסוגלת לאתר 8-אוקסו-dGuo בדגימות ביולוגיות. אימות נוספת היא מעבר להיקף של מאמר זה. עם זאת, אנו בטוחים כי פרוטוקול זה יעזור לי פוטנציאלימשתמשים לקבוע את המידה שבה הם צריכים לאמת באופן רשמי בפרוטוקול, בהתאם לצרכיהם. רשימה של צעדים הנדרשים לאימות הפורמאלית של השיטה מסופקת נוספת. במהלך הפיתוח והפריסה של שיטה לזיהוי 8-אוקסו-dGuo, שיטות שפורסמו נסקרו ומאוחדת. לכן, שיטה זו מבטלת את הצורך לאסוף מידע ממספר מקורות שפורסמו, כי לעתים קרובות חסרי פרטים ניסיוניים חשובים גם בעת מתן אמצעי מהיר ופשוט של בדיקות אם השיטה לאיתור והכימות של 8-אוקסו-dGuo כבר אימצה בהצלחה. שיטה מותאמת זה הועסקה לנתח בהצלחה דגימות DNA מהתאים בתרבית רקמה ועכברית. מאמר זה וידאו יסייע קבוצות אחרות בהקמת שיטה יעילה לגילוי אמין וכימות של 8-אוקסו-dGuo ידי HPLC-ED.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>8-oxo-dGuo standard</td>
			<td>Cayman Chemical Company</td>
			<td>89320</td>
			<td>Inappropriately referred to as &#34;8-hydroxy-2&#39;-deoxy Guanosine&#34; - see Fig. 1 and text for details</td>
		</tr>
		<tr>
			<td>Alkaline phosphatase&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>P5931</td>
			<td>From E.coli</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 with e.g. 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&#160;</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

סטרס חמצונים קשור הרבה תהליכים פיסיולוגיים ופתולוגיים, כמו גם חילוף חומרים xenobiotic, מוביל לחמצון של Biomacromolecules, כולל DNA. לכן, זיהוי יעיל של חמצון ה- DNA הוא חשוב עבור מגוון רחב של תחומי מחקר, כוללים רפואה וטוקסיקולוגיה. סמן ביולוגי משותף של דנ&quot;א פגום oxidatively הוא 8-אוקסו-7,8-Dihydro-2&#39;-deoxyguanosine (8-אוקסו-dGuo; לעתים קרובות מכונה בטעות כ8-הידרוקסי-2&#39;deoxyguanosine (8-OH-dGuo או 8 -oxo-DG)). כמה פרוטוקולים ל8-אוקסו-dGuo מדידה על ידי כרומטוגרפיה נוזלית בלחץ גבוה עם זיהוי אלקטרוכימי (HPLC-ED) תוארו. עם זאת, אלה יושמו בעיקר ל- DNA מטוהר שטופל בפרו-חמצון. בנוסף, עקב הבדלים מתודולוגיים בין מעבדות, בעיקר בשל הבדלים בציוד אנליטי, האימוץ של שיטות שפורסמו לצורך זיהוי של 8-אוקסו-dGuo ידי HPLC-ED דורש אופטימיזציה זהירה בכל מעבדה.פרוטוקול מקיף, המתאר תהליך האופטימיזציה כזה, חסר. כאן, פרוטוקול מפורט מתואר לגילוי של 8-אוקסו-dGuo ידי HPLC-ED, בDNA מהתאים בתרבית או רקמות של בעלי חיים. זה מדגים כיצד הכנת דגימת DNA יכולה להיות מותאמת בקלות ובמהירות כדי למזער חמצון ה- DNA לא רצוי שיכול להתרחש במהלך הכנת מדגם. פרוטוקול זה מראה כיצד לזהות 8-אוקסו-dGuo בתאים בתרבית אדם אדנוקרצינומה מכתשי (כלומר, תאי A549) שטופלו בסוכן חמצון KBrO 3, ומהטחול של עכברים שנחשפו לdibenzo פחמימנים ארומטיים polycyclic (def, עמ &#39;) chrysene (DBC, שנקרא בעבר פיר dibenzo (, יב), DalP). בסך הכל, עבודה זו מדגימה כיצד מתודולוגיה HPLC-ED יכולה להיות מותאמת בקלות לזיהוי של 8-אוקסו-dGuo בדגימות ביולוגיות.

dGuo נצפה ליש זמן שמירה של 4.7 דקות ואילו 8-אוקסו-dGuo זמן שמירה של כ 6.4 דקות (איור 2 א &#39;וב&#39;). יש הבדל כ -1,000 פי בגבה השיא בין שני analytes, כפי שניתן לראות באיור 2 ג. Voltammograms ל8-אוקסו-dGuo וdGuo התקבלו על ידי סטנדרטים פועלים בפוטנציאל עבודה בטווח של 0.2-1.1 V. פוטנציאל העב?...

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

המטרה של פרוטוקול זה היא הגילוי של סמן חמצון ה- DNA, 8-אוקסו-7,8-Dihydro-2&#39;-deoxyguanosine (8-אוקסו-dGuo) על ידי HPLC-ED, בDNA מהתאים בתרבית או רקמות של בעלי חיים.

HPLC המדידה של ה- DNA חמצון ביומרקר, 8-אוקסו-7,8-Dihydro-2&#39;-deoxyguanosine, בתאים בתרבית ורקמות בעלי החיים

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

hplc-measurement-dna-oxidation-biomarker-8-oxo-78-dihydro-2

Research

JoVE Journal

Chemistry

26.2K Views.  Health Canada.  המטרה של פרוטוקול זה היא הגילוי של סמן חמצון ה- DNA, 8-אוקסו-7,8-Dihydro-2'-deoxyguanosine (8-אוקסו-dGuo) על ידי HPLC-ED, בDNA מהתאים בתרבית או רקמות של בעלי חיים. 

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

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.

מדידת מצב יציב, טרום מצב יציב, וחד מחזור קינטי לפעילות ה-DNA Glycosylase

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).

מתקפל ואפיון של רובוט ביו-תגובה מה- DNA אוריגמי

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

Construction of Models for Nondestructive Prediction of Ingredient Contents in Blueberries by Near-infrared Spectroscopy Based on HPLC Measurements

Nondestructive prediction of ingredient contents of farm products is useful to ship and sell the products with guaranteed qualities. Here, near-infrared spectroscopy is used to predict nondestructively total sugar, total organic acid, and total anthocyanin content in each blueberry. The technique is expected to enable the selection of only delicious blueberries from all harvested ones. The near-infrared absorption spectra of blueberries are measured with the diffuse reflectance mode at the positions not on the calyx. The ingredient contents of a blueberry determined by high-performance liquid chromatography are used to construct models to predict the ingredient contents from observed spectra. Partial least squares regression is used for the construction of the models. It is necessary to properly select the pretreatments for the observed spectra and the wavelength regions of the spectra used for analyses. Validations are necessary for the constructed models to confirm that the ingredient contents are predicted with practical accuracies. Here we present a protocol to construct and validate the models for nondestructive prediction of ingredient contents in blueberries by near-infrared spectroscopy.

We present here a protocol to construct and validate models for nondestructive prediction of total sugar, total organic acid, and total anthocyanin content in individual blueberries by near-infrared spectroscopy.

בניית מודלים לחיזוי הורס של תוכן מרכיב האוכמניות ידי ספקטרוסקופיה האינפרה-אדום הקרוב בהתבסס על מדידות HPLC

Nondestructive prediction of ingredient contents of farm products is useful to ship and sell the products with guaranteed qualities. Here, near-infrared ...

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.

HPLC מבוססי Assay לפקח תאיים נוקלאוטיד / מטבוליזם nucleoside בתאי לוקמיה לימפוציטית כרונית

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.

סינתזה של חלבון Bioconjugates<em&gt; דרך</em&gt; כימיה ציסטאין-maleimide

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.

קביעת ההרכב הכימי של ממשקי קורוזיה מונעים / מתכת עם XPS: חמצון טבילת הודעת מזעור

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.

ייצור ובדיקה של מדי חום פוטוני

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.

המדידה בו זמנית HDAC1 ופעילות HDAC6 הלה תאים באמצעות 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 ...

DNA-magnetic Particle Binding Analysis by Dynamic and Electrophoretic Light Scattering

Isolation of DNA using magnetic particles is a field of high importance in biotechnology and molecular biology research. This protocol describes the evaluation of DNA-magnetic particles binding via dynamic light scattering (DLS) and electrophoretic light scattering (ELS). Analysis by DLS provides valuable information on the physicochemical properties of particles including particle size, polydispersity, and zeta potential. The latter describes the surface charge of the particle which plays major role in electrostatic binding of materials such as DNA. Here, a comparative analysis exploits three chemical modifications of nanoparticles and microparticles and their effects on DNA binding and elution. Chemical modifications by branched polyethylenimine, tetraethyl orthosilicate and (3-aminopropyl)triethoxysilane are investigated. Since DNA exhibits a negative charge, it is expected that zeta potential of particle surface will decrease upon binding of DNA. Forming of clusters should also affect particle size. In order to investigate the efficiency of these particles in isolation and elution of DNA, the particles are mixed with DNA in low pH (~6), high ionic strength and dehydration environment. Particles are washed on magnet and then DNA is eluted by Tris-HCl buffer (pH = 8). DNA copy number is estimated using quantitative polymerase chain reaction (PCR). Zeta potential, particle size, polydispersity and quantitative PCR data are evaluated and compared. DLS is an insightful and supporting method of analysis that adds a new perspective to the process of screening of particles for DNA isolation.

This protocol describes the synthesis of magnetic particles and evaluation of their DNA-binding properties via dynamic and electrophoretic light scattering. This method focuses on monitoring changes in particle size, their polydispersity and zeta potential of particle surface which play major role in binding of materials such as DNA.

חלקיקים מגנטיים-DNA מחייב ניתוח לפי פיזור אור דינאמי, Electrophoretic

Isolation of DNA using magnetic particles is a field of high importance in biotechnology and molecular biology research. This protocol describes the ...

Disentangling High Strength Copolymer Aramid Fibers to Enable the Determination of Their Mechanical Properties

Traditionally, soft body armor has been made from poly(p-phenylene terephthalamide) (PPTA) and ultra-high molecular weight polyethylene. However, to diversify the fiber choices in the United States body armor market, copolymer fibers based on the combination of 5-amino-2-(p-aminophenyl) benzimidazole (PBIA) and the more conventional PPTA were introduced. Little is known regarding the long-term stability of these fibers, but as condensation polymers, they are expected to have potential sensitivity to moisture and humidity. Therefore, characterizing the strength of the materials and understanding their vulnerability to environmental conditions is important for evaluating their use lifetime in safety applications. Ballistic resistance and other critical structural properties of these fibers are predicated on their strength. To accurately determine the strength of the individual fibers, it is necessary to disentangle them from the yarn without introducing any damage. Three aramid-based copolymer fibers were selected for the study. The fibers were washed with acetone followed by methanol to remove an organic coating that held the individual fibers in each yarn bundle together. This coating makes it difficult to separate single fibers from the yarn bundle for mechanical testing without damaging the fibers and affecting their strength. After washing, fourier transform infrared (FTIR) spectroscopy was performed on both washed and unwashed samples and the results were compared. This experiment has shown that there are no significant variations in the spectra of poly(p-phenylene-benzimidazole-terephthalamide-co-p-phenylene terephthalamide) (PBIA-co-PPTA1) and PBIA-co-PPTA3 after washing, and only a small variation in intensity for PBIA. This indicates that the acetone and methanol rinses are not adversely affecting the fibers and causing chemical degradation. Additionally, single fiber tensile testing was performed on the washed fibers to characterize their initial tensile strength and strain to failure, and compare those to other reported values. Iterative procedural development was necessary to find a successful method for performing tensile testing on these fibers.

The primary goal of the study is to develop a protocol to prepare consistent specimens for accurate mechanical testing of high strength copolymer aramid fibers, by removing a coating and disentangling the individual fiber strands without introducing significant chemical or physical degradation.

Disentangling חוזק גבוה קופולימר ארמיד סיבים כדי לאפשר קביעת תכונות מכניות שלהם

Traditionally, soft body armor has been made from poly(p-phenylene terephthalamide) (PPTA) and ultra-high molecular weight polyethylene. However, to ...