Name: a colorimetric method for measuring iron content in plants
Uploaded: 2018-09-07T13:30:00
Description: Watch this Scientific Journal Video about A Colorimetric Method for Measuring Iron Content in Plants at JoVE.com

Diese Arbeit wurde durch einen Zuschuss aus dem Hauptamt des israelischen Ministeriums für Landwirtschaft (#16-16-0003) von Israel Ministerium für Wissenschaft, Technologie und Spaceand unterstützt.

(1) Pflanzenmaterial und Wachstumsbedingungen
<ol><li>Samen einer Tabak (Sorte Samsun) Samen pro Topf 5 x 5 cm mit standard Topf Medium gefüllt. Stellen Sie die Töpfe auf Tabletts. Wachsen Sie die Pflanzen im Wachstum Zimmer unter anstrengenden Bedingungen (16/8 h hell/dunkel) bei einer konstanten Temperatur von 23 ° C. Spülen Sie mit Wasser aus dem Wasserhahn bis Wasser fließt aus dem Topf.</li>
	<li>Beginnen Sie nach 50±5 Tagen Fe Behandlungen in der Bewässerung, entsprechend die Konzentrationen geeignet für ...

<ol>
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T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Arabidopsis+defensin+gene,+AtPDF1.1,+mediates+defence+against+Pectobacterium+carotovorum+subsp.+carotovorum+via+an+iron-withholding+defence+system.">The Arabidopsis defensin gene, AtPDF1.1, mediates defence against Pectobacterium carotovorum subsp. carotovorum via an iron-withholding defence system.</a> Science Reports. 7 (1), 1-14 (2017).</li><li>Johnson, D. B., Kanao, T., Hedrich, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Redox+transformations+of+iron+at+extremely+low+pH:+Fundamental+and+applied+aspects.">Redox transformations of iron at extremely low pH: Fundamental and applied aspects.</a> Frontiers in Microbiology. 3, 1-13 (2012).</li><li>Stumm, W., Lee, G. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxygenation+of+ferrous+iron.">Oxygenation of ferrous iron.</a> Industrial &amp; Engineering Chemistry. 53 (2), 143-146 (1961).</li><li>Jones, A. M., Griffin, P. J., Collins, R. N., Waite, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ferrous+iron+oxidation+under+acidic+conditions+-+The+effect+of+ferric+oxide+surfaces.">Ferrous iron oxidation under acidic conditions - The effect of ferric oxide surfaces.</a> Geochimica et Cosmochimica Acta. 145, 1-12 (2014).</li><li>Hawkesworth, C. J., Kemp, A. I. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+of+the+continental+crust.">Evolution of the continental crust.</a> Nature. 443 (7113), (2006).</li><li>Thompson, L. M., Louis, M., Troeh, F. R., Thompson, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Soils and soil fertility. , (1973).</li><li>Krueger, B. J., Grassian, V. H., Cowin, J. P., Laskin, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneous+chemistry+of+individual+mineral+dust+particles+from+different+dust+source+regions:+The+importance+of+particle+mineralogy.">Heterogeneous chemistry of individual mineral dust particles from different dust source regions: The importance of particle mineralogy.</a> Atmospheric Environment. 38 (36), 6253-6261 (2004).</li><li>Bewick, V., Cheek, L., Ball, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Statistics+review+7:+Correlation+and+regression.">Statistics review 7: Correlation and regression.</a> Journal of Critical Care. 7 (6), 451-459 (2003).</li><li>Asuero, A. G., Sayago, A., Gonz&#225;lez, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+correlation+coefficient:+An+overview.">The correlation coefficient: An overview.</a> Critical Reviews in Analytical Chemistry. 36 (1), 41-59 (2006).</li><li>. Analytical Chemistry. Calibration Curves Available from: <a target="_blank" href="https://www.jove.com/science-education/10188/calibration-curves">https://www.jove.com/science-education/10188/calibration-curves</a> (2018)</li></ol>

Eisen-Messung im Pflanzengewebe ist sehr wichtig für die Bewertung der Auswirkungen der Bewässerung oder andere Umweltbedingungen. Wir hier eine einfache und genaue kolorimetrischen Verfahren zur Fe Content Messung in Tabakblättern, die ohne weiteres auf andere Pflanzenarten und Gewebe angepasst werden können.
Bedingungen für die kolorimetrischen Verfahren zu optimieren, haben wir einen niedrigen pH-Wert Medium (pH < 1.0) um Eisen Löslichkeit zu ermöglichen. Der Brennvorgang wurde durch...

Eisen (Fe) ist ein wichtiger Mikronährstoff in allen lebenden Organismen. In Pflanzen ist es ein wesentlicher Mikronährstoff-1 wegen seiner Beteiligung an grundlegenden Prozesse, wie Atmung, Photosynthese und Chlorophyll Biosynthese. Hohe Ansammlung von freien Eisenionen ist schädlich für die Pflanzenzellen durch Reaktionen führt zur Freisetzung von freien Radikalen verursacht oxidativen Stress. Um Eisen in der Pflanzenzelle Homöostase sind Ionen in Vakuolen gespeichert und innerhalb Ferritins, Protein Käfige direkt beteiligt sind Eisen-Homöostase-2 und der wichtigsten Speicherstruktur von Eisen in allen lebenden Organismen abgesondert. Zur gleichen Zeit betrifft Eisenmangel Anämie einen Großteil der menschlichen Bevölkerung, was zu einem steigenden Bedarf an Anlage Fe biofortifikation. Aufgrund der einzigartigen Eigenschaften der Pflanze Ferritin bietet Essen Anreicherung mit Ferritin-Eisen eine vielversprechende Strategie zur Bekämpfung dieses Problems der Unterernährung3.
Eisen-Ionen sind hauptsächlich gefunden in zwei Oxidationsstufen, nämlich dem Eisen (zweiwertigen Fe2 + oder Eisen (II)) und Eisen (dreiwertiges Fe3 + oder Eisen (III)) Formen. Verschiedene andere Formen von Eisen, z. B. Eisen-Cluster4, finden sich auch in den Zellen. FE wird gespeichert als Eisenoxid innerhalb der Zelle und natürlich Formen Hematites (Fe2O3) und Ferryhidrites ((Fe3 +)2O3•0.5 H2O) unter physiologischen Bedingungen5. Die hydroxide gebildet in diesen Reaktionen, vor allem Eisen Form, haben sehr geringe Löslichkeit. Eisen-Aufbewahrung richtet sich daher nach den pH-Wert der Lösung und ist weitgehend in einen festen Zustand über pH 5-6.
Angesichts der schlechten Löslichkeit und hohe Reaktivität der Fe muss die Übertragung unter den pflanzlichen Geweben und Organen geeignete Komplexbildner Moleküle zugeordnet werden. Darüber hinaus müssen die Redox-Staaten zwischen dem Eisen und Eisen Formen1 gesteuert werden. In Blätter etwa 80 % des Eisens findet sich in photosynthetischen Zellen durch seine wesentliche Rollen in das elektronentransportsystem, bei der Biosynthese von Zellfarbstoffe, Chlorophyll und anderen Häm-Moleküle, und bei der Bildung von Fe-S-Cluster7. Im Falle von Eisen Übermaß innerhalb der Zelle ist der Überschuss in die Vakuole umgesiedelt, wo das Metall in Ferritin-Moleküle8gespeichert ist.
Eisen kann durch mehrere Methoden, einschließlich Flamme Atom-Absorptions-Spektroskopie9 (FAAS) oder farbmetrische Assays10, das ehemalige sein viel präziser als die letzteren in Pflanzengeweben gemessen werden. FAAS ist eine hochpräzise Technik, mit der man die elementare Zusammensetzung einer Probe auf der Grundlage der elektromagnetischen Emissionen der einzelnen Elemente bestimmen kann. FAAS konvertiert Metallionen in Zustände durch Flamme erhitzen der Probe, was zu Ionen-Anregung und Emission einer bestimmten Wellenlänge, wenn eine bestimmte Ion in seinem Grundzustand zurückkehrt. Die Emissionen aus den verschiedenen Ionen sind getrennt durch einen Monochromator und durch Absorption Sensor11erkannt. FAAS dient somit direkt Eisen-Konzentrationen zu quantifizieren. Andere Techniken zur Visualisierung von Eisen in biologischen Geweben sind jedoch verfügbar. Induktiv-gekoppelte Plasma-Massenspektrometrie (ICP-MS)12 ist eine sehr präzise Technik zur Messung von Eisen und anderen Spurenelementen, sondern der Mangel an Ausrüstung, sowohl für FAAS und ICP-MS, ist ein häufiges Problem. Auf der anderen Seite Eisen Messung von Thiocyanat Farbmetrik13 nicht präzise genug und nicht geringe Schwankungen zwischen Proben erkennt. Preußischen blaue Färbung14,15,16,17 ist eine indirekte Methode basiert auf der Reaktion von dreiwertigem Kaliumferrocyanid (K4Fe(CN)6) mit Fe-kationen, produzieren ein starke blaue Farbe, und dient zur qualitativen Eisen-Erkennung in histologischen Abschnitten von tierischen und pflanzlichen Geweben.
Metallic (null-Valent) Eisen ist selten in der Lithosphäre. Die dominierende nicht komplexiert Ionische Form des Eisens in der Umgebung richtet sich vor allem durch die Menge an Sauerstoff in der Umgebung mit Eisen Eisen als relativ häufiger in anoxischen Umgebungen und dreiwertigem Eisen überwiegt in aeroben Websites. Diese letztere Form ist auch in extrem sauren Umgebungen, dominierend, obwohl die Erreger der eisenhaltige Eisenoxidation oft in anoxischen und sauren Umgebung18unterscheiden. Wenn Eisen ist in 4 % solubilisiert HCl (pH 0) in einer aeroben Umgebung, der Großteil der verdünnten Eisen vorhanden ist, als das Eisen (Fe3 +)19,20bilden.
Die Reaktionen zwischen Fe-Ionen und K4Fe(CN)6 lauten wie folgt:
FE3 +: FeCl3 + K4Fe(CN)6 = KFe(III)Fe(II)(CN)6¯ + 3KCl
FE2 +: 4 FeCl2 + 2 K4Fe(CN)6 Fe4(Fe(CN)6)2 + 8 = KCl
In der vorliegenden Studie fragten wir ob preußischen Blaufärbung zur Messung der Eisengehalt in Lösung nützlich sein kann.
Wir überprüft zunächst die Korrelation zwischen der Konzentration von Fe in wässriger Lösung und preußischen Blaufärbung. Die Fe (als FeCl2, FeCl3 oder ein 1:1 Mischung aus beidem) Konzentration in wässrigen Lösungen wurde sowohl durch Atomspektroskopie Extinktion (OD) nach Zugabe von Preußischblau gemessen. Abbildung 1 zeigt die lineare Regression-Kurven für Messungen von jeder Methode. Wir festgestellt, dass die preußische blaue Methode zur quantitativen Analyse von Eisen-Konzentration in der Lösung verwendet werden kann.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57408/57408fig1.jpg"> Abbildung 1: lineare Regressionen zwischen Fe-Konzentration gemessen an FAAS und leichte Extinktion (OD, 715 nm) durch die preußische blau-Methode erhalten. Die blauen Quadrate und Linie stellen die Fe2 + Lösung dar, die roten Quadrate und Linie repräsentieren die Fe3 + Lösung und die schwarzen Quadrate und Linie repräsentieren eine 1:1 Mischung aus Fe2 + und Fe3 +. Die folgenden Regressionen wurden erzielt: [Fe2 +] = 3 + 123 X OD, R = 0.996, R2 = 0.989; [Fe3 +] = 1 + 292 X OD, R = 0,999, R2 = 0.997; und [Fe2 + / 3 +] = 11 + 146 X OD, R = 0.983, R2 = 0.956. Fe2 + Spender war FeCl2 und Fe3 + Spender war FeCl3. <a href="https://www.jove.com/files/ftp_upload/57408/57408fig1large.jpg" target="_blank">Bitte klicken Sie hier für eine größere Version dieser Figur.</a>
Um die farbmetrischen preußische blau Methode für quantitative Eisen Analyse von Pflanzengewebe anzupassen, war der Eisengehalt von Tabak Blatt Asche gemessen Flamme Absorption Atomspektroskopie und preußischen Blaufärbung. Es gab gute Korrelation zwischen den Ergebnissen aus durch die beiden Techniken.

<table>
	<tbody>
		<tr>
			<td>Potassium Hexacyanoferrate(II)</td>
			<td>Fisher Chemical</td>
			<td>14459-95-1</td>
			<td>Reagent for the Pussian Blue</td>
		</tr>
		<tr>
			<td>Millex Syringe Filter Unit, Vial Vent 0.22 &#956;m</td>
			<td>Millec</td>
			<td>SLGP033RS</td>
			<td>Filter used to filter the ashes + 4% HCl Solution</td>
		</tr>
		<tr>
			<td>Scintillation Vials</td>
			<td>Fisherbrand</td>
			<td>03-337-4</td>
			<td>Used to keep the dry powdered plant material during the burning procedure.</td>
		</tr>
		<tr>
			<td>Disposable Syringe 10 ml</td>
			<td>Medi-Plus</td>
			<td>1931</td>
			<td>Syringe used during the filtration</td>
		</tr>
		<tr>
			<td>Hydrochloric acid</td>
			<td>Sigma-Aldrich</td>
			<td>231-595-7</td>
			<td>Used in the 4% HCl solution to dilute the ashes and clean the materials</td>
		</tr>
		<tr>
			<td>Tobacco, Nicotiana tabacum cv. Samsun NN</td>
			<td>Obtained from Prof. Simon Barak and routinely used in the Zaccai Lab</td>
			<td>Barak S, Nejidat A, Heimer Y, Volokita M. Transcriptional and posttranscriptional regulation of the glycolate oxidase gene in tobacco seedlings. Plant Molecular Biology. 2001 Mar 1;45(4):399-407.</td>
			<td>Tobacco cultivar used in this protocol</td>
		</tr>
		<tr>
			<td>Glass Wool (Rock Wool)</td>
			<td>Sigma-Aldrich</td>
			<td>659997-17-3</td>
			<td>Used in the procedure of burning samples in the furnace.</td>
		</tr>
	</tbody>
</table>

a colorimetric method for measuring iron content in plants

Eisen, eines der wichtigsten Mikronährstoffe in lebenden Organismen, engagiert sich in grundlegenden Prozesse, wie Atmung und Photosynthese. Gehalt an Eisen ist in allen Organismen, in Höhe von ca. 0,009 % Trockensubstanz in Pflanzen eher gering. Bis heute ist eine der genauesten Methoden zur Messung der Eisenkonzentration im Pflanzengewebe Flamme Absorption Atomspektroskopie. Aber dieser Ansatz ist zeitaufwändig und teuer und erfordert speziellen Geräte, die in Anlage Labors nicht häufig zu finden. Daher braucht man eine einfachere, aber genaue Methode, die routinemäßig eingesetzt werden kann. Die farbmetrische Preußischblau Methode wird regelmäßig für qualitative Eisen Färbung in Tier- und histologischen Abschnitte verwendet. In dieser Studie haben wir die Preußischblau Methode zur quantitativen Messungen des Eisens im Tabak Blätter angepasst. Wir überprüft die Genauigkeit dieser Methode mit Atomspektroskopie und Preußischblau beflecken, zum Messen der Eisengehalt in den gleichen Proben und fand eine lineare Regression (R2 = 0.988) zwischen den beiden Verfahren. Wir schlussfolgern, dass die Preußischblau Methode zur quantitativen Eisen-Messung im Pflanzengewebe präzise, einfach und kostengünstig ist. Die lineare Regression, die hier vorgestellten möglicherweise jedoch nicht geeignet für andere Pflanzenarten, wegen möglicher Wechselwirkungen zwischen der Probe und Reagenz. Einrichtung einer Regression Kurve benötigt somit für verschiedene Pflanzenarten.

Wenn dieses Protokoll korrekt durchgeführt wird, sollte man ausgezeichnete Korrelation zwischen den Ergebnissen der preußischen blau und atomare Spektroskopie-Methoden. Daher die preußische blaue Methode leicht lässt sich eine genaue Messung der Eisenkonzentration in Pflanzenproben, wie Sie sich in das folgende Experiment.
Tabakpflanzen waren gewachsen, wie im Protokoll beschrieben und bewässert mit Wasser, die verschiedene...

Watch this Scientific Journal Video about A Colorimetric Method for Measuring Iron Content in Plants at JoVE.com

A Colorimetric Method for Measuring Iron Content in Plants

Ein kolorimetrischen Verfahren zur Messung der Eisengehalt in Pflanzen

Wir präsentieren Ihnen ein einfaches und zuverlässiges Protokoll zur Messung der Eisengehalt im Pflanzengewebe mit der kolorimetrischen Preußischblau-Methode.

Iron, one of the most important micronutrients in living organisms, is involved in basic processes, such as respiration and photosynthesis. Iron content ...

Just medically it can help answer a key question in biology field. Such as how can we easily quantify iron in biology core samples. The main venture of this technique is that it's a easy to produce, and a very precise colorimetric technique.While preferring this technique, you should avoid iron contamination. Visual demonstration of this method will help you to understand its different steps. To begin this procedure, prepare five centimeter by five centimeter pots by filling them with standard pot medium.Plant one tobacco seed in each pot. Transfer the plants to a growth room that is at a constant temperature, under long day conditions of 23 degrees Celsius. Irrigate with tap water until water drains from the pot, and grow the plants for approximately 50 days.After this time, start iron treatments in the irrigation at concentrations appropriate for the experiment. Irrigate the plants with this solution every two days for six to eight days. Then, detach the leaves from the stem by hand, making sure not to use any metal equipment.Using a spray bottle filled with double distilled water, clean each leaf. Dry the leaves on a paper towel, and then transfer them into a paper bag. Place the leaf filled bags in an oven, set to a constant temperature of 80 degrees Celsius, for two to three days.Clean the mortar and pestle twice, with a 4%hydrochloric acid solution. And use a filter paper to dry. Using the mortar and pestle, crush the dry leaves to a powder.Then, transfer the powder to sterile 15 milliliter plastic tubes. First, weigh a new, sealed 20 milliliter scintillation vial without its lid. Either note the weight or tare the scale.Then add the crushed leaves. Weigh the sample in the vial, and note the weight. Use rock wool to close the vial.Weigh three additional vials without adding samples, for use as controls, making note of each weight. Next, transfer the sample and control vials to a furnace. Burn the leaves as outlined in the text protocol.After this, let the samples cool to approximately 100 degrees Celsius. Using heavy gloves and tweezers, remove the samples from the furnace, making sure to hold the vial exteriorly. Place the vials on a flat surface.Remove the rock wool, and close the vials with their original lids. Then, weigh the three control vials, and calculate their average weight gain. If the weight gain is equal to or above 1%of the ash weight, use this value as an estimate of the measurement error.Weigh a 15 milliliter plastic tube. Either record the weight or tare the scale. Then transfer the ashes to the tube.Record this value, which is the ash weight. Next, add five milliliters of the one molar hydrochloric acid solution to the ashes. Filter the ashes through a 22 micrometer filter.Then add an additional five milliliters of the one molar hydrochloric acid solution through the same filter, leading to a final sample volume of 10 milliliters. After this, remove four milliliters from each sample for measurement by atomic spectroscopy. And determine the iron concentration per gram of ash as outlined in the text protocol.To begin, add four grams of potassium ferrocyanide to 100 milliliters of double distilled water, to prepare the Prussian blue solution. Vortex to mix, and store at 4 degrees Celsius until ready to use. When ready to use, mix 50 milliliters of Prussian blue solution with 50 milliliters of one molar hydrochloric acid to serve as the blank solution.Then, use a pipette to mix 0.5 milliliters of a previously obtained sample, with 0.5 milliliters of Prussian blue solution. Let this mixture rest for at least one minute, but for less than five minutes. Transfer this mixture to a cuvette, and use a spectrophotometer to measure the optical density at 715 nanometers.Then, determine the optical density per gram of ash, and plot the linear regression between the iron concentrations, as outlined in the text protocol. The representative results from 21 tobacco leaf samples show that the iron concentration in the irrigation water greatly affected the iron content in the leaf. The spectra of all 21 representative samples are then tested with the Prussian blue method.As seen here, the absorbance at 715 nanometers is the optimal wavelength while measuring solutions containing different concentrations of iron two and iron three. A linear regression curve, which is obtained by plotting iron concentration values obtained by atomic spectroscopy, versus the absorbance values, obtained by the Prussian blue method, allows for new samples of the same plant type to be analyzed. Don't forget that working with HCL can be extremely hazardous.And precaution, such as eye protection, should always be taken while performing this procedure. In the linear regression, it's important to verify that the results are in a realistic range. After its development, this technique can help biology researcher explore additional plants for food production or bioremediation.

a-colorimetric-method-for-measuring-iron-content-in-plants

Research

JoVE Journal

Biochemistry

Covalent Binding of Antibodies to Cellulose Paper Discs and Their Applications in Naked-eye Colorimetric Immunoassays

This report presents two methods for the covalent immobilization of capture antibodies on cellulose filter paper grade No. 1 (medium-flow filter paper) discs and grade No. 113 (fast-flow filter paper) discs. These cellulose paper discs were grafted with amine functional groups through a silane coupling technique before the antibodies were immobilized on them. Periodate oxidation and glutaraldehyde cross-linking methods were used to graft capture antibodies on the cellulose paper discs. In order to ensure the maximum binding capacity of the capture antibodies to their targets after immobilization, the effects of various concentrations of sodium periodate, glutaraldehyde, and capture antibodies on the surface of the paper discs were investigated. The antibodies that were coated on the amine-functionalized cellulose paper discs through a glutaraldehyde cross-linking agent showed enhanced binding activity to the target when compared to the periodate oxidation method. IgG (in mouse reference serum) was used as a reference target in this study to test the application of covalently immobilized antibodies through glutaraldehyde. A new paper-based, enzyme-linked immunosorbent assay (ELISA) was successfully developed and validated for the detection of IgG. This method does not require equipment, and it can detect 100 ng/ml of IgG. The fast-flow filter paper was more sensitive than the medium-flow filter paper. The incubation period of this assay was short and required small sample volumes. This naked-eye, colorimetric immunoassay can be extended to detect other targets that are identified with conventional ELISA.

In this manuscript, periodate oxidation and glutaraldehyde cross-linking methods for covalent immobilization of antibodies on paper discs are presented. The binding activity of immobilized antibodies was evaluated. Based on these results, a glutaraldehyde cross-linking method was used to develop a novel paper-based immunoassay for immunoglobulin G (IgG) detection.

Kovalente Bindung von Antikörpern an Cellulose Papierscheiben und ihre Anwendungen in der Naked-Augen Kolorimetrische Immunoassays

This report presents two methods for the covalent immobilization of capture antibodies on cellulose filter paper grade No. 1 (medium-flow filter paper) ...

Forschung

Biochemie

A Method for Measuring RNA N6-methyladenosine Modifications in Cells and Tissues

N6-Methyladenosine (m6A) modifications of RNA are diverse and ubiquitous amongst eukaryotes. They occur in mRNA, rRNA, tRNA, and microRNA. Recent studies have revealed that these reversible RNA modifications affect RNA splicing, translation, degradation, and localization. Multiple physiological processes, like circadian rhythms, stem cell pluripotency, fibrosis, triglyceride metabolism, and obesity are also controlled by m6A modifications. Immunoprecipitation/sequencing, mass spectrometry, and modified northern blotting are some of the methods commonly employed to measure m6A modifications. Herein, we present a northeastern blotting technique for measuring m6A modifications. The current protocol provides good size separation of RNA, better accommodation and standardization for various experimental designs, and clear delineation of m6A modifications in various sources of RNA. While m6A modifications are known to have a crucial impact on human physiology relating to circadian rhythms and obesity, their roles in other (patho)physiological states are unclear. Therefore, investigations on m6A modifications have immense possibility to provide key insights into molecular physiology.

A Method for Measuring RNA N6-methyladenosine Modifications in Cells and Tissues

A modified northern blotting method for measuring N6-methyladenosine (m6A) modifications in RNA is described. The current method can detect modifications in diverse RNAs and controls under various experimental designs.

Verfahren zum Messen von RNA<em&gt; N</em<sup&gt; 6</sup&gt; -methyladenosine Änderungen in Zellen und Geweben

N6-Methyladenosine (m6A) modifications of RNA are diverse and ubiquitous amongst eukaryotes. They occur in mRNA, rRNA, tRNA, and microRNA. Recent studies ...

A Method for Measuring Metabolism in Sorted Subpopulations of Complex Cell Communities Using Stable Isotope Tracing

Mammalian cell types exhibit specialized metabolism, and there is ample evidence that various co-existing cell types engage in metabolic cooperation. Moreover, even cultures of a single cell type may contain cells in distinct metabolic states, such as resting or cycling cells. Methods for measuring metabolic activities of such subpopulations are valuable tools for understanding cellular metabolism. Complex cell populations are most commonly separated using a cell sorter, and subpopulations isolated by this method can be analyzed by metabolomics methods. However, a problem with this approach is that the cell sorting procedure subjects cells to stresses that may distort their metabolism.
To overcome these issues, we reasoned that the mass isotopomer distributions (MIDs) of metabolites from cells cultured with stable isotope-labeled nutrients are likely to be more stable than absolute metabolite concentrations, because MIDs are formed over longer time scales and should be less affected by short-term exposure to cell sorting conditions. Here, we describe a method based on this principle, combining cell sorting with liquid chromatography-high resolution mass spectrometry (LC-HRMS). The procedure involves analyzing three types of samples: (1) metabolite extracts obtained directly from the complex population; (2) extracts of "mock sorted" cells passed through the cell sorter instrument without gating any specific population; and (3) extracts of the actual sorted populations. The mock sorted cells are compared against direct extraction to verify that MIDs are indeed not altered by the cell sorting procedure itself, prior to analyzing the actual sorted populations. We show example results from HeLa cells sorted according to cell cycle phase, revealing changes in nucleotide metabolism.

This article describes a method for studying cellular metabolism in complex communities of multiple cell types, using a combination of stable isotope tracing, cell sorting to isolate specific cell types, and mass spectrometry.

Ein Verfahren zur Messung des Metabolismus in sortierter Subpopulationen von komplexen Zell Gemeinschaften Verwendung stabiler Isotopen-Tracing

Mammalian cell types exhibit specialized metabolism, and there is ample evidence that various co-existing cell types engage in metabolic cooperation. ...

Multimer-PAGE: A Method for Capturing and Resolving Protein Complexes in Biological Samples

There are many well-developed methods for purifying and studying single proteins and peptides. However, most cellular functions are carried out by networks of interacting protein complexes, which are often difficult to investigate because their binding is non-covalent and easily perturbed by purification techniques. This work describes a method of stabilizing and separating native protein complexes from unmodified tissue using two-dimensional polyacrylamide gel electrophoresis. Tissue lysate is loaded onto a non-denaturing blue-native polyacrylamide gel, then an electric current is applied until the protein migrates a short distance into the gel. The gel strip containing the migrated protein is then excised and incubated with the amine-reactive cross-linking reagent dithiobis(succinimidyl propionate), which covalently stabilizes protein complexes. The gel strip containing cross-linked complexes is then cast into a sodium dodecyl sulfate polyacrylamide gel, and the complexes are separated completely. The method relies on techniques and materials familiar to most molecular biologists, meaning it is inexpensive and easy to learn. While it is limited in its ability to adequately separate extremely large complexes, and has not been universally successful, the method was able to capture a wide variety of well-studied complexes, and is likely applicable to many systems of interest.

A method for stabilizing and separating native protein complexes from unmodified tissue lysate using an amine-reactive protein cross-linker coupled to a novel two-dimensional polyacrylamide gel electrophoresis (PAGE) system is presented.

Multimer-PAGE: Ein Verfahren zum Erfassen und Auflösen von Proteinkomplexen in biologischen Proben

There are many well-developed methods for purifying and studying single proteins and peptides. However, most cellular functions are carried out by ...

Measuring Protein Binding to F-actin by Co-sedimentation

Filamentous actin (F-actin) organization within cells is regulated by a large number of actin-binding proteins that control actin nucleation, growth, cross-linking and/or disassembly. This protocol describes a technique &#8211; the actin co-sedimentation, or pelleting, assay &#8211; to determine whether a protein or protein domain binds F-actin and to measure the affinity of the interaction (i.e., the dissociation equilibrium constant). In this technique, a protein of interest is first incubated with F-actin in solution. Then, differential centrifugation is used to sediment the actin filaments, and the pelleted material is analyzed by SDS-PAGE. If the protein of interest binds F-actin, it will co-sediment with the actin filaments. The products of the binding reaction (i.e., F-actin and the protein of interest) can be quantified to determine the affinity of the interaction. The actin pelleting assay is a straightforward technique for determining if a protein of interest binds F-actin and for assessing how changes to that protein, such as ligand binding, affect its interaction with F-actin.

This protocol describes a method to test the ability of a protein to co-sediment with filamentous actin (F-actin) and, if binding is observed, to measure the affinity of the interaction.

Messung der Proteinbindung an F-Aktin durch Co-Sedimentation

Filamentous actin (F-actin) organization within cells is regulated by a large number of actin-binding proteins that control actin nucleation, growth, ...

Determination of the Glycogen Content in Cyanobacteria

Cyanobacteria accumulate glycogen as a major intracellular carbon and energy storage&#160;during photosynthesis. Recent developments in research have highlighted complex mechanisms of glycogen metabolism, including the diel cycle of biosynthesis and catabolism, redox regulation, and the involvement of non-coding RNA. At the same time, efforts are being made to redirect carbon from glycogen to desirable products in genetically engineered cyanobacteria to enhance product yields. Several methods are used to determine the glycogen contents in cyanobacteria, with variable accuracies and technical complexities. Here, we provide a detailed protocol for the reliable determination of the glycogen content in cyanobacteria that can be performed in a standard life science laboratory. The protocol entails the selective precipitation of glycogen from the cell lysate and the enzymatic depolymerization of glycogen to generate glucose monomers, which are detected by a glucose oxidase-peroxidase (GOD-POD) enzyme coupled assay. The method has been applied to Synechocystis sp. PCC 6803 and Synechococcus sp. PCC 7002, two model cyanobacterial species that are widely used in metabolic engineering. Moreover, the method successfully showed differences in the glycogen contents between the wildtype and mutants defective in regulatory elements or glycogen biosynthetic genes.

Here, we present a reliable and easy assay to measure the glycogen content in cyanobacterial cells. The procedure entails precipitation, selectable depolymerization, and the detection of glucose residues. This method is suitable for both wildtype and genetically engineered strains and can facilitate the metabolic engineering of cyanobacteria.

Bestimmung des Glykogengehalts in Cyanobakterien

Cyanobacteria accumulate glycogen as a major intracellular carbon and energy storage&#160;during photosynthesis. Recent developments in research have ...

Structural Characterization of Mannan Cell Wall Polysaccharides in Plants Using PACE

Plant cell wall polysaccharides are notoriously difficult to analyze, and most methods require expensive equipment, skilled operators, and large amounts of purified material. Here, we describe a simple method for gaining detailed polysaccharide structural information, including resolution of structural isomers. For polysaccharide analysis by gel electrophoresis (PACE), plant cell wall material is hydrolyzed with glycosyl hydrolases specific to the polysaccharide of interest (e.g., mannanases for mannan). Large format polyacrylamide gels are then used to separate the released oligosaccharides, which have been fluorescently labeled. Gels can be visualized with a modified gel imaging system (see Table of Materials). The resulting oligosaccharide fingerprint can either be compared qualitatively or, with replication, quantitatively. Linkage and branching information can be established using additional glycosyl hydrolases (e.g., mannosidases and galactosidases). Whilst this protocol describes a method for analyzing glucomannan structure, it can be applied to any polysaccharide for which characterized glycosyl hydrolases exist. Alternatively, it can be used to characterize novel glycosyl hydrolases using defined polysaccharide substrates.

A protocol for the structural analysis of polysaccharides by gel electrophoresis (PACE), using mannan as an example, is described.

Strukturelle Charakterisierung von Mannan Zellwand Polysaccharide in Anlagen, in denen Tempo

Plant cell wall polysaccharides are notoriously difficult to analyze, and most methods require expensive equipment, skilled operators, and large amounts ...

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages for identifying biologically active chemical structures that can modify complex phenotypes such as lifespan. Described here is a simple protocol for producing hundreds of 96-well culture plates with fairly consistent numbers of C. elegans in each well. Next, we specified how to use these cultures to screen thousands of chemicals for effects on the lifespan of the nematode C. elegans. This protocol makes use of temperature sensitive sterile strains, agar plate conditions, and simple animal handling to facilitate the rapid and high throughput production of synchronized animal cultures for screening.

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

Here we describe a simple protocol for rapidly producing hundreds of nematode growth media agar, 96-well culture plates with consistent numbers of Caenorhhabditis elegans per well. These cultures are useful for the phenotypic screening of whole organisms. We focus here on using these cultures to screen chemicals for pro-longevity effects.

Eine einfache Methode zur chemischen Hochdurchsatz-Screening in Caenorhabditis Elegans

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages ...

Spectrophotometric Determination of Phycobiliprotein Content in Cyanobacterium Synechocystis

This is a simple protocol for the quantitative determination of phycobiliprotein content in the model cyanobacterium Synechocystis. Phycobiliproteins are the most important components of phycobilisomes, the major light-harvesting antennae in cyanobacteria and several algae taxa. The phycobilisomes of Synechocystis contain two phycobiliproteins: phycocyanin and allophycocyanin. This protocol describes a simple, efficient, and reliable method for the quantitative determination of both phycocyanin and allophycocyanin in this model cyanobacterium. We compared several methods of phycobiliprotein extraction and spectrophotometric quantification. The extraction procedure as described in this protocol was also successfully applied to other cyanobacteria strains such as Cyanothece sp., Synechococcuselongatus, Spirulina sp., Arthrospira sp., and Nostoc sp., as well as to red algae Porphyridium cruentum. However, the extinction coefficients of specific phycobiliproteins from various taxa can differ and it is, therefore, recommended to validate the spectrophotometric quantification method for every single strain individually. The protocol requires little time and can be performed in any standard life science laboratory since it requires only standard equipment.

Spectrophotometric Determination of Phycobiliprotein Content in Cyanobacterium Synechocystis

Here, we present a protocol to quantitatively determine phycobiliprotein content in the cyanobacterium Synechocystis using a spectrophotometric method. The extraction procedure was also successfully applied to other cyanobacteria and algae strains; however, due to variations in pigment absorption spectra, it is necessary to test the spectrophotometric equations for each strain individually.

Photometrische Bestimmung des Cyanobakteriums Synechocystis Phycobiliprotein Inhalt

This is a simple protocol for the quantitative determination of phycobiliprotein content in the model cyanobacterium Synechocystis. Phycobiliproteins are ...

Measuring and Interpreting Oxygen Consumption Rates in Whole Fly Head Segments

Regulated metabolic activity is essential for the normal functioning of living cells. Indeed, altered metabolic activity is causally linked with the progression of cancer, diabetes, neurodegeneration, and aging to name a few. For instance, changes in mitochondrial activity, the cell&#39;s metabolic powerhouse, have been characterized in many such diseases. Generally, the oxygen consumption rates of mitochondria were considered a reliable readout of mitochondrial activity and measurements in some of these studies were based on isolated mitochondria or cells. However, such conditions may not represent the complexity of a whole tissue. Recently, we have developed a novel method that enables the dynamic measurement of oxygen consumption rates from whole isolated fly heads. By utilizing this method, we have recorded lower oxygen consumption rates of the whole head segment in young versus aged flies. Secondly, we have discovered that lysine deacetylase inhibitors rapidly alter the oxygen consumption in the whole head. Our novel technique may therefore aid in uncovering new properties of various drugs, which may impact metabolic rates. Furthermore, our method may give a better understanding of metabolic behavior in an experimental setup that more closely resembles physiological states.

Measuring alterations in metabolic rates is central to understanding the progression of various diseases and aging. Here, we present a novel technique to measure whole head oxygen consumption that more closely resembles the physiological state and may aid in revealing novel drugs that modify mitochondrial activity.

Messung und Interpretation von Sauerstoff-Verbrauch im ganzen fliegen Kopf Segmente

Regulated metabolic activity is essential for the normal functioning of living cells. Indeed, altered metabolic activity is causally linked with the ...