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

Dette arbeidet ble støttet av Israels departementet for vitenskap, teknologi og Spaceand av et stipend fra vitenskapsmann for Israels departementet av landbruket (#16-16-0003).

1. plante materiale og vekst betingelser
<ol><li>Frø en tobakk (sorten Samsun) frø per 5 cm x 5 cm pott fylt med standard potten medium. Plass pottene på skuffer. Dyrke planter i et vekst rom under lang dag forhold (16/8 h lys/mørke) ved en konstant temperatur på 23 ° C. Vanne med vann inntil vannet faller ut fra potten.</li>
	<li>Etter 50±5 dager, start Fe behandlinger i vanning, etter konsentrasjonene egnet for eksperimentet. For eksempel brukte vi en rekke konsentrasjoner av jern fra 0 til 6 mM, supplert med ...

<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. 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Jern måling i anlegget vev er svært viktig for å vurdere effekten av vanning eller andre miljøforhold. Her beskrev vi en enkel og nøyaktig kolorimetrisk metode for Fe innhold måling i tobakksblader som kan være lett tilpasses andre plantearter og vev.
Optimalisere betingelser for metoden kolorimetrisk, brukte vi et medium for lav pH (pH < 1.0) for å tillate jern oppløselighet. Den brennende prosessen ble utført å løslate alle former av jern og for å sikre at ingen forurensninger t...

Iron (Fe) er en viktig micronutrient i alle levende organismer. I planter er det en viktig micronutrient1 på grunn av sitt engasjement i basic prosesser, for eksempel åndedrett, fotosyntese og klorofyll biosyntesen. Høy opphopning av ledig jern ioner er skadelig til anlegget celler skyldes reaksjoner fører til utgivelsen av frie radikaler forårsake oksidativt stress. For å opprettholde jern homeostase i anlegget celle, ioner lagres i vacuoles og sequestered i ferritins, protein burene direkte involvert i iron homeostase2 og den primære lagringsområdet strukturen i jern i alle levende organismer. Samtidig påvirker jernmangel anemi en betydelig andel av befolkningen, som resulterer i et økende behov for plante Fe biofortification. På grunn av de unike egenskapene til anlegget ferritin tilbyr mat berikelse med ferritin-jern en lovende strategi for å bekjempe dette problemet av underernæring3.
Jern ioner er hovedsakelig finnes i to oksidasjon stater, nemlig den jern (divalent Fe2 + eller jern (II)) og ferric (trivalent Fe3 + eller jern (III)) skjemaer. Flere andre former av jern, som jern klynger4, finnes også i celler. Fe lagres som jernoksid i cellen og naturlig former hematites (Fe2O3) og ferryhidrites ((Fe3 +)2O3•0.5 H2O) under fysiologiske forhold5. Hydroxides i disse reaksjonene, spesielt ferric skjemaet har svært lav oppløselighet. Jern oppbevaring påvirkes derfor av pH av løsningen og er i stor grad et solid state over pH 56.
Vurderer dårlig løslighet og høy reaktivitet Fe, må overtatt blant anlegget vev og organer være forbundet med egnet chelaterande molekyler. Videre må sine redoks tilstander mellom jern og ferric skjemaer1 kontrolleres. I blader, rundt 80% av jern finnes i fotosynteseaktiviteten celler, på grunn av dens viktige roller i elektronet transportsystem, i biosyntesen av cytochromes, klorofyll og andre måltema molekyler, og i dannelsen av Fe-S klynger7. Ved jern overflødig i cellen, er overskuddet translocated i vacuole der metall lagres i ferritin molekyler8.
Jern kan måles i anlegget vev av flere metoder, inkludert flamme atomic absorpsjon spektroskopi9 (FAAS) eller kolorimetrisk analyser10, det tidligere er langt mer presis enn sistnevnte. FAAS er en svært nøyaktige teknikk som gjør det mulig å bestemme elementær sammensetningen av et eksempel på grunnlag av elektromagnetiske utslipp av enkeltelementer. FAAS konverterer metall ioner å Atom stater av flamme-oppvarming av prøven, fører til ion eksitasjon og utslipp av en bestemt bølgelengde når en gitt ion tilbake til tilstanden bakken. Utslippene fra de ulike ionene er atskilt med en monokromator og oppdaget av en absorpsjon sensor11. FAAS bidrar dermed til direkte kvantifisere konsentrasjoner av jern. Andre teknikker for å visualisere jern i biologisk vev er imidlertid tilgjengelige. Induktivt kombinert plasma-masse spektroskopi (ICP-MS)12 er en veldig presis teknikk for måling av jern og andre sporstoffer, men mangel på utstyr, både FAAS og ICP-MS, er et vanlig problem. På den annen side, jern måling av thiocyanate colorimetry13 mangler presisjon og svikter å merker små variasjoner mellom eksempler. Prøyssiske blå flekker14,15,16,17 er en indirekte metode basert på reaksjonen av kalium ferric ferrocyanide (K4Fe(CN)6) med Fe kasjoner, produsere en sterk blå farge, og brukes for kvalitativ jern gjenkjenning histologiske deler av dyre- og plantelivet vev.
Metallisk (null-valent) jern er sjeldne i jordskorpen. Dominerende ikke-kompleksbundet ionisk form av jern i miljøet er stort sett diktert av mengden av oksygen i omgivelsene, med jern jern er relativt mer rikelig i anoksisk miljøer og ferric jern predominating i aerobic områder. Denne siste formen er også dominerende i svært sure miljøer, selv om den forårsaker agenter av jern jern oksidasjon ofte avvike anoksisk og sure18. Når jern er solubilized i 4% HCl (pH 0) i aerobic miljø, av utvannet jern finnes som den ferric danner (Fe3 +)19,20.
Reaksjonene mellom Fe ioner og K4Fe(CN)6 er som følger:
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
Studien spurte vi om prøyssiske blå flekker kan være nyttig for å måle jern nivåer i løsningen.
I utgangspunktet bekreftet vi sammenhengen mellom konsentrasjonen av Fe i vandig løsning og prøyssiske blå flekker. Fe (som FeCl2, FeCl3 eller en 1:1 blanding av to) konsentrasjonen i vandige løsninger ble målt etter atomic spektroskopi og absorbansen (OD) etter prøyssiske blå. Figur 1 viser lineær regresjon kurvene for målinger ved hver metode. Vi konkluderte med at den prøyssiske blå metoden kan brukes for kvantitativ analyse av jern konsentrasjon i løsningen.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57408/57408fig1.jpg"> Figur 1: lineære regresjoner mellom Fe konsentrasjon målt ved FAAS og lys absorbansen (OD, 715 nm) innhentet av Prussian blå metoden. Den blå firkanter og linjen representerer Fe2 + løsningen, røde firkanter og linjen representerer Fe3 + løsningen og de svarte firkantene og linje representerer en 1:1 mellom Fe2 + og Fe3 +. Følgende regresjoner anskaffet: [Fe2 +] = 3 + 123 x OD, r = 0.996, R2 = 0.989; [Fe3 +] = 1 + 292 x OD, r = 0.999, R2 = 0.997; og [Fe2 / 3 +] = 11 + 146 x OD, r = 0.983, R2 = 0.956. Fe2 + donor var FeCl2 Fe3 + donor var FeCl3. <a href="https://www.jove.com/files/ftp_upload/57408/57408fig1large.jpg" target="_blank">Klikk her for å se en større versjon av dette tallet.</a>
For å tilpasse kolorimetrisk prøyssiske blå metoden for kvantitative jern analyse av anlegget vev, ble jerninnhold på tobakk blad asken målt ved flamme absorpsjon atomic spektroskopi og prøyssiske blå flekker. Det var god sammenheng mellom resultatene av de to teknikkene.

<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

Jern, en av de viktigste mikronæringsstoffer i levende organismer, er involvert i grunnleggende prosesser, som åndedrett og fotosyntese. Jerninnhold er ganske lav i alle organismer, pålydende i planter ca 0,009% av tørrvekt. Hittil er en av de mest nøyaktige metodene for måling av jern konsentrasjon i anlegget vev flamme absorpsjon atomic spektroskopi. Men dette er tidkrevende og kostbar og krever bestemt utstyr ikke ofte funnet i anlegget laboratorier. Derfor er en enklere, men presis metode som kan brukes rutinemessig nødvendig. Kolorimetrisk prøyssiske blå metoden brukes jevnlig for kvalitativ jern farging i dyre- og plantelivet histologiske deler. I denne studien tilpasset vi den prøyssiske blå metoden for kvantitative mål av jern i tobakk blader. Vi validert nøyaktigheten av denne metoden bruker både Atom spektroskopi og prøyssiske blå flekker å måle jerninnhold i samme prøvene og fant en lineær regresjon (R2 = 0.988) mellom de to prosedyrene. Vi konkludere med at den prøyssiske blå metoden for kvantitative jern måling i anlegget vev er nøyaktig, enkel og rimelig. Men kan lineær regresjon presenteres her ikke være passende for andre plantearter, på grunn av potensielle interaksjoner mellom prøven og reagens. Etablering av en regresjons-kurven er derfor nødvendig for forskjellige plantearter.

Når denne protokollen utføres riktig, bør man få utmerket sammenheng mellom resultatene av Prussian blå og Atom spektroskopi metoder. Derfor kan metoden prøyssiske blå lett brukes til å få en nøyaktig måling av jern konsentrasjon i anlegget prøver, som gjenspeiles i følgende eksperimentet.
Tobakk planter dyrket som beskrevet i protokollen og vannet med vann som inneholder forskjellige jern konsentrasjoner (0, 1, 2, ...

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

Kolorimetrisk metode for å måle jerninnhold i planter

Vi presenterer en enkel og pålitelig protokoll for å måle jerninnhold anlegget vev med metoden for kolorimetrisk prøyssiske blå.

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.

Kovalent Binding av antistoffer til cellulosepapir plater og deres applikasjoner i Naked-eye Kolorimetriske Immunoassays

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

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.

En metode for måling av RNA<em&gt; N</em<sup&gt; 6</sup&gt; -methyladenosine Endringer i celler og vev

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.

En metode for å måle metabolismen i sortert subpopulasjoner av komplekse Cell Communities Bruke stabile isotoper 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: En metode for å fange og løse protein komplekser i biologiske prøver

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.

Måling av proteinbinding til F-aktin ved co-sedimentering

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.

Bestemmelse av glykogeninnholdet i cyanobakterier

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 karakteristikk av Mannan cellevegg polysakkarider i planter ved hjelp av 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.

En enkel metode for høy gjennomstrømning kjemiske Screening i 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.

Spektrofotometri fastsettelse av Phycobiliprotein innhold i Cyanobacterium Synechocystis

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.

Måling og tolke oksygen forbruk priser helt Fly hodet segmenter

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