وقد تم تمويل هذا البحث من قبل مبادرة وزارة الصحة الكندية الجينوم بحوث والتنمية (GRDI) والاستراتيجية التنظيمية الكندية للتكنولوجيا الحيوية (CRSB). الكتاب ليس لديهم تضارب في المصالح.

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No conflict of interest or competing financial interests declared.

على الرغم من أن 8 أوكسو dGuo تم الإبلاغ عن العلامات البيولوجية مفيدة للأكسدة الحمض النووي، يمكن تقدير لها موثوق تحديا. على الرغم من أن العديد من الطرق التي نشرت موجودة، وهناك حاجة ل، نظرة عامة وصفية شاملة للبروتوكول للسماح للباحثين لنشر الأسلوب في مختبراتهم. نحن هنا تق?...

تساهم أنواع الاكسجين التفاعلية (ROS)، الذي مستويات ثابتة للدولة يمكن أن تزيد خلال العديد من الحالات المرضية والتمثيل الغذائي xenotoxic، إلى زيادة وتيرة الحمض النووي من التلف التأكسدي. بين عدة النووية nucleobases الممكنة منتجات الأكسدة، ويمكن أن الحمض النووي من التلف التأكسدي بسهولة أن تقاس باستخدام علامة مستقرة 8 أوكسو 7،8-ثنائي هيدرو-2&#39;-deoxyguanosine (8 أوكسو dGuo)، التي تعد واحدة من أشكال المؤكسد من 2 &#39; -deoxyguanosine (dGuo) 1. 8-أوكسو dGuo هو آفة DNA الأكثر وفرة 2، وبالتالي، وقد درس لمزيد من التفاصيل والعلامات البيولوجية أكسدة الحمض النووي على الرغم من وجود الحمض النووي عدة منتجات الأكسدة 3. في البشر، وهذا الضرر يمكن إصلاحه عن طريق قاعدة إصلاح الختان بنسبة 8-oxoguanine glycosylase 1 (hOGG1) 4. إذا تركت دون إصلاح، ويمكن 8 أوكسو dGuo المساهمة في تشكيل قاعدة الطفرات الزوج إحلال (أي G إلى T transversions) 4. الأهم من ذلك، 8 أوكسو dGuo هو علامة المنشأة FOالحمض النووي من التلف (ص) في ما يتعلق استهلال وتعزيز التسرطن 2. ولذلك التقدير الدقيق 8 أوكسو dGuo هو العلامات البيولوجية مفيدة ومرغوبة من الحمض النووي من التلف التأكسدي 5. هناك ارتباك واسع النطاق في الأدبيات المتعلقة الأسماء الصحيحة لأشكال تلف oxidatively ل2-deoxyguanosine وعلاوة على ذلك، والاسم الصحيح للمجمع (ق) تقاس بشكل روتيني والعلامات البيولوجية من الحمض النووي من التلف التأكسدي 6. من 6،8-diketo و 6 إنول، 8-كيتوني أشكال صنواني من 8 أوكسو dGuo (كما هو موضح في الشكل رقم 1) هما tautomers أبرز مناقشتها في الأدب 5،7. شكل 6،8-diketo هو الشكل الأبرز في درجة الحموضة الفسيولوجية 7.4، و هو الأبرز أكسدة الحمض النووي المنتج 7. لذا، 8 أوكسو dGuo، بدلا من 8 هيدروكسي dGuo هو الاسم الأنسب لهذا المنتج أكسدة 6. ومن المهم أيضا أن نلاحظ أن 2-deoxyguanosine (dGuo)، بدلا من nucleobبورصة عمان جوانين (غواتيمالا) أو غوانوزين ريبونوكليوزيد (قوه) على التوالي، تم الكشف من قبل معظم وسائل 6. كشف دقيق وتقدير من 8 أوكسو dGuo يمثل تحديا للأسباب التالية: أ) التباين في الهضم من عينة من الحمض النووي، والثاني) الأكسدة عرضية من dGuo إلى 8 أوكسو dGuo التي يمكن أن تحدث أثناء إعداد العينات، والثالث) الحاجة للتحقق من صحة الفعال للالتحليلي طريقة HPLC-ED 8. في هذا البروتوكول، ونحن تهدف إلى تحقيق ط) من خلال توفير الظروف المواتية لاستكمال عملية الهضم DNA والثاني) من قبل خالب إدراج المعدن وحلول المعالجة خالب وكاشف عزل DNA الخاص، في حين أن الثالث) كانت موجهة جزئيا فقط إدراج الضوابط الإيجابية، وبالتالي تنص على أن الأسلوب هو قادر على اكتشاف 8 أوكسو dGuo في العينات البيولوجية. مزيد من التأكيد هو أبعد من نطاق هذه الورقة. ومع ذلك، فإننا واثقون من أن هذا البروتوكول سوف تساعد المحتملينالمستخدمين على تحديد المدى الذي يحتاجون إليه للتحقق من صحة رسميا البروتوكول، حسب أغراضها. وهناك قائمة من الخطوات المطلوبة للمصادقة رسمية من طريقة أخرى. خلال تطوير ونشر طريقة للكشف عن 8 أوكسو dGuo، تم استعراض أساليب نشر وتوحيدها. وبالتالي، فإن هذا الأسلوب يلغي الحاجة إلى جمع المعلومات من عدة مصادر منشورة التي تفتقر في كثير من الأحيان تفاصيل التجريبية مهمة بينما توفر أيضا وسيلة سريعة ومباشرة من اختبار إذا تم اعتماد أسلوب للكشف وتقدير من 8 أوكسو dGuo بنجاح. كان يعمل هذا الأسلوب تكييفها لتحليل عينات من الحمض النووي بنجاح من الخلايا المستزرعة والأنسجة الفئران. هذه المقالة الفيديو سوف تساعد مجموعات أخرى في إنشاء وسيلة فعالة للكشف عن موثوقة وتقدير من 8 أوكسو dGuo من قبل 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

ويرتبط الاكسدة مع العديد من العمليات الفسيولوجية والمرضية، وكذلك التمثيل الغذائي أجنبي بيولوجيا، مما يؤدي إلى أكسدة الجزيئات الكبيرة، بما في ذلك الحمض النووي. ولذلك، كشف الفعال للأكسدة الحمض النووي مهم لمجموعة متنوعة من التخصصات البحثية، بما في ذلك الأدوية والسموم. A العلامات البيولوجية المشتركة للأضرار الحمض النووي oxidatively هو 8-أوكسو 7،8-ثنائي هيدرو-2&#39;-deoxyguanosine (8 أوكسو dGuo، غالبا ما يشار إليها باسم خطأ 8-هيدروكسي-2&#39;-deoxyguanosine (8-OH-dGuo أو 8 -oxo-DG)). وقد وصفت عدة بروتوكولات لقياس 8 أوكسو dGuo ارتفاع ضغط اللوني السائل مع كشف الكهروكيميائية (HPLC-ED). ومع ذلك، طبقت هذه أساسا لDNA تنقية تعامل مع الموالية للتأكسد. بالإضافة إلى ذلك، بسبب الاختلافات المنهجية بين المختبرات، ويرجع ذلك أساسا إلى الاختلافات في المعدات التحليلية، واعتماد أساليب نشرت للكشف عن 8 أوكسو dGuo من قبل HPLC-ED يتطلب التحسين دقيق من قبل كل مختبر. Aبروتوكول شامل، واصفا مثل هذه العملية الأمثل، غير متوفرة. هنا، يتم وصف بروتوكول مفصلة للكشف عن 8 أوكسو dGuo من قبل HPLC-ED، في DNA من الخلايا المستنبتة أو الأنسجة الحيوانية. فإنه يوضح كيفية إعداد عينة من الحمض النووي يمكن بسهولة وبسرعة الأمثل لتقليل أكسدة الحمض النووي غير المرغوب فيها التي يمكن أن تحدث أثناء إعداد العينات. ويظهر هذا البروتوكول كيفية اكتشاف 8 أوكسو dGuo في الخلايا المستزرعة الإنسان غدية السنخية (أي الخلايا A549) تعامل مع عامل مؤكسد KBrO 3، ومن الطحال من الفئران المعرضة للثنائي بنزو الهيدروكربونات العطرية متعددة الحلقات (صفر، ع) chrysene (DBC، المعروف سابقا باسم بنزو (أ، ل) بيرين، DalP). عموما، يوضح هذا العمل كيف منهجية HPLC-ED يمكن أن يكون الأمثل بسهولة للكشف عن 8 أوكسو dGuo في العينات البيولوجية.

ولوحظ dGuo أن يكون لها الوقت الاحتفاظ 4.7 دقيقة في حين كان 8 أوكسو dGuo فترة الاحتفاظ حوالي 6.4 دقيقة (الشكل 2A وباء). هناك اختلاف حول 1000 أضعاف في مرتفعات الذروة بين التحاليل اثنين، كما يظهر في الشكل 2C. تم الحصول على Voltammograms لمدة 8-أوكسو dGuo وdGuo وفقا للمعايير الت...

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

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

والهدف من هذا البروتوكول هو الكشف عن الحمض النووي الأكسدة علامة، 8 أوكسو 7،8-ثنائي هيدرو-2&#39;-deoxyguanosine (8 أوكسو dGuo) من قبل HPLC-ED، في DNA من الخلايا المستنبتة أو الأنسجة الحيوانية.

HPLC قياس الحمض النووي الأكسدة العلامات البيولوجية، 8 أوكسو 7،8-ثنائي هيدرو-2&#39;-deoxyguanosine، في الخلايا المستزرعة والأنسجة الحيوانية

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

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

26.1K Views.  Health Canada.  والهدف من هذا البروتوكول هو الكشف عن الحمض النووي الأكسدة علامة، 8 أوكسو 7،8-ثنائي هيدرو-2'-deoxyguanosine (8 أوكسو dGuo) من قبل HPLC-ED، في DNA من الخلايا المستنبتة أو الأنسجة الحيوانية. 

Video: HPLC Measurement of the DNA Oxidation Biomarker, 8-oxo-7,8-dihydro-2&#8217;-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.

ثابت للدولة، قبل المطرد للدولة، واحدة دوران قياس الحركية للنشاط الحمض النووي Glycosylase

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

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.

القائم HPLC الفحص لمراقبة خارج الخلية النكليوتيد / نوكليوزيد الأيض في خلايا سرطان الدم الليمفاوي المزمن الإنسان

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

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.

تحديد التركيب الكيميائي لالمانع للتآكل واجهات / المعادن مع XPS: التقليل المشاركة الغمر الأكسدة

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.

تصنيع واختبار الحرارة الضوئية

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.

قياس واحد من HDAC1 والنشاط HDAC6 في الحلة الخلايا باستخدام أوهبلك--مرض التصلب العصبي المتعدد

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.

الحمض النووي-المغناطيسي الجسيمات ربط التحليل بتشتت الضوء الديناميكي والغرواني الكهربي

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.

مقدر ألياف الاراميد كوبوليمر قوة عالية لتمكين تحديد خواصها الميكانيكية

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

Technical Aspect of the Automated Synthesis and Real-Time Kinetic Evaluation of [11C]SNAP-7941

Positron emission tomography (PET) is an essential molecular imaging technique providing insights into pathways and using specific targeted radioligands for in vivo investigations. Within this protocol, a robust and reliable remote-controlled radiosynthesis of [11C]SNAP-7941, an antagonist to the melanin-concentrating hormone receptor 1, is described. The radiosynthesis starts with cyclotron produced [11C]CO2 that is subsequently further reacted via a gas-phase transition to [11C]CH3OTf. Then, this reactive intermediate is introduced to the precursor solution and forms the respective radiotracer. Chemical as well as the radiochemical purity are determined by means of RP-HPLC, routinely implemented in the radiopharmaceutical quality control process. Additionally, the molar activity is calculated as it is a necessity for the following real-time kinetic investigations. Furthermore, [11C]SNAP-7941 is applied to MDCKII-WT and MDCKII-hMDR1 cells for evaluating the impact of P-glycoprotein (P-gp) expression on cell accumulation. For this reason, the P-gp expressing cell line (MDCKII-hMDR1) is either used without or with blocking prior to experiments by means of the P-gp substrate (&#177;)-verapamil and the results are compared to the ones observed for the wildtype cells. The overall experimental approach demonstrates the importance of a precise time-management that is essential for every preclinical and clinical study using PET tracers radiolabelled with short-lived nuclides, such as carbon-11 (half-life: 20 min).

Technical Aspect of the Automated Synthesis and Real-Time Kinetic Evaluation of [11C]SNAP-7941

Here, we represent a protocol for the fully automated radiolabelling of [11C]SNAP-7941 and the analysis of the real-time kinetics of this PET-tracer on P-gp expressing and non-expressing cells.

الجانب التقني من التوليف الآلي والتقييم الحركي في الوقت الحقيقي من [11C] SNAP-7941

Positron emission tomography (PET) is an essential molecular imaging technique providing insights into pathways and using specific targeted radioligands ...

Quantitative Atomic-Site Analysis of Functional Dopants/Point Defects in Crystalline Materials by Electron-Channeling-Enhanced Microanalysis

A novel elemental and chemical analysis scheme based on electron-channeling phenomena in crystalline materials is introduced, where the incident high-energy electron beam is rocked with the submicrometric pivot point fixed on a specimen. This method enables us to quantitatively derive the site occupancies and site-dependent chemical information of impurities or intentionally doped functional elements in a specimen, using energy-dispersive X-ray spectroscopy and electron energy-loss spectroscopy attached to a scanning transmission electron microscope, which is of significant interest to current materials science, particularly related to nanotechnologies. This scheme is applicable to any combination of elements even when the conventional Rietveld analysis by X-ray or neutron diffraction occasionally fails to provide the desired results because of limited sample sizes and close scattering factors of neighboring elements in the periodic table. In this methodological article, we demonstrate the basic experimental procedure and analysis method of the present beam-rocking microanalysis.

We provide a general outline of quantitative microanalysis methods for estimating the site occupancies of impurities and their chemical states by taking advantage of electron-channeling phenomena under incident electron beam-rocking conditions, which reliably extract information from minority species, light elements, oxygen vacancies, and other point/line/planar defects.

تحليل الموقع الذري الكمي لعيوب Dopants / نقطة وظيفية في المواد البلورية عن طريق التحليل الدقيق المعزز لتوجيه الإلكترون

A novel elemental and chemical analysis scheme based on electron-channeling phenomena in crystalline materials is introduced, where the incident ...

HPLC Coupled with Chemical Fingerprinting for Multi-Pattern Recognition for Identifying the Authenticity of Clematidis Armandii Caulis

A method for identifying Chinese medicinal materials and their related adulterants was constructed by taking Clematidis Armandii Caulis (Chuanmutong, a universally used traditional Chinese medicine) as an example. Ten batches of genuine Chuanmutong varieties and five batches of related adulterants were analyzed and compared based on the high-performance liquid chromatography (HPLC) fingerprints combined with chemometrics, including cluster analysis (CA), principal component analysis (PCA), and orthogonal partial least-squares discrimination analysis (OPLS-DA). In addition, the content of &#946;-sitosterol was determined. The control chemical fingerprint of Chuanmutong was established, and 12 common peaks were identified. The similarity between the fingerprint of 10 batches of genuine Chuanmutong varieties and the control fingerprint was 0.910-0.989, while the similarity of five batches of adulterants was only 0.133-0.720. Based on the common peaks in the chromatogram, 15 batches of samples were classified into three content levels by PCA, and were aggregated into four categories by CA, achieving a clear distinction between authentic Chuanmutong and adulterants of Chuanmutong. Further, seven differential components that can effectively identify authentic Chuanmutong and adulterants of Chuanmutong were found through OPLS-DA. The &#946;-sitosterol content of 10 batches of genuine Chuanmutong varieties was 97.53-161.56 &#181;g/g, while the &#946;-sitosterol content of the five batches of adulterants varied greatly, among which the &#946;-sitosterol content of Clematis peterae Hand.-Mazz. and Clematis gouriana Roxb. Var. finetii Rehd. et Wils. was significantly lower than that of authentic varieties of Chuanmutong. The HPLC index component content and chemical fingerprint multi-pattern recognition method established in this study provide a new strategy for effectively identifying authentic Chinese medicinal materials and related adulterants.

HPLC Coupled with Chemical Fingerprinting for Multi-Pattern Recognition for Identifying the Authenticity of Clematidis Armandii Caulis

Here, we present a protocol to establish high-performance liquid chromatography (HPLC), coupled with chemical fingerprint multi-pattern recognition, which provides a new strategy for effectively identifying the genuine varieties of Clematidis Armandii Caulis and its adulterants.

HPLC إلى جانب البصمات الكيميائية للتعرف على الأنماط المتعددة لتحديد أصالة ياسمين أرماندي كوليس

A method for identifying Chinese medicinal materials and their related adulterants was constructed by taking Clematidis Armandii Caulis (Chuanmutong, a ...