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

Questo lavoro è stato supportato da l'Israele Ministero della scienza, tecnologia e progettuali da una sovvenzione del Chief Scientist del Ministero israeliano dell'agricoltura (n. 16-16-0003).

1. materiale e le condizioni di crescita della pianta
<ol><li>Semi di semi di tabacco di una (cultivar Samsun) per vaso di 5cm x 5cm riempito con mezzo vaso standard. Posizionare i vasi su vassoi. Crescere le piante in una stanza di crescita in condizioni di giorno lungo (16/8 h chiaro/scuro) ad una temperatura costante di 23 ° C. Irrigare con acqua di rubinetto fino a quando gli scoli dell'acqua dalla pentola.</li>
	<li>Dopo giorni di 50 ± 5, iniziare trattamenti di Fe nell'irrigazione, secondo le concentrazioni ada...

<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. 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Misurazione di ferro nei tessuti vegetali è molto importante per la valutazione degli effetti di irrigazione o altre condizioni ambientali. Qui, abbiamo descritto un metodo colorimetrico facile e preciso per la misurazione del contenuto Fe in foglie di tabacco, che può essere facilmente adattato ad altre specie di piante e tessuti.
Nell'ottimizzazione delle condizioni per il metodo colorimetrico, abbiamo usato un mezzo a basso pH (pH < 1.0) per consentire la solubilità del ferro. Il process...

Ferro (Fe) è un micronutriente importante in tutti gli organismi viventi. Nelle piante, è un micronutriente essenziale1 a causa del suo coinvolgimento in processi di base, come la respirazione, la fotosintesi e la clorofilla biosintesi. Elevato accumulo di ioni di ferro libero è nociva alla pianta cellule a causa di reazioni che portano alla liberazione di radicali liberi che causano stress ossidativo. Per mantenere l'omeostasi del ferro all'interno della cellula vegetale, gli ioni sono memorizzati nei vacuoli e sequestrati all'interno di ferritine, gabbie di proteina direttamente coinvolte nell' omeostasi di ferro2 e la struttura di archiviazione principale del ferro in tutti gli organismi viventi. Allo stesso tempo, l'anemia sideropenica colpisce una percentuale significativa della popolazione umana, risultante in un crescente bisogno di pianta Fe bioarricchimento. A causa delle proprietà uniche di ferritina pianta, arricchimento di cibo con ferritina-ferro offre una strategia promettente per combattere questo problema della malnutrizione3.
Gli ioni di ferro sono principalmente trovati in due stati di ossidazione, vale a dire i metalli ferrosi (ferro (II) o bivalente Fe2 + ) e ferrico (ferro (III) o trivalente Fe3 + ) forme. Diverse altre forme di ferro, come ferro cluster4, si trovano anche nelle cellule. Fe è memorizzato come ossido di ferro all'interno della cella e naturalmente ematiti forme (Fe2O3) e ferryhidrites ((Fe3 +)2O30,5 H2O) sotto condizioni fisiologiche5. Idrossidi formate in queste reazioni, soprattutto la forma ferrica, hanno solubilità molto bassa. Ritenzione di ferro conseguenza è influenzata dal pH della soluzione ed è in gran parte in uno stato solido sopra pH 56.
Considerando la scarsa solubilità e l'alta reattività del Fe, suo trasferimento tra organi e tessuti vegetali deve essere associato a molecole chelanti adatte. Inoltre, i suoi Stati redox tra i moduli ferrosi e ferrici1 devono essere controllati. All'interno di foglie, circa l'80% del ferro si trova nelle cellule fotosintetiche, a causa di suoi ruoli essenziali del sistema di trasporto dell'elettrone, nella biosintesi dei citocromi, clorofilla e altre molecole di eme e nella formazione di Fe-S cluster7. Nel caso di ferro in eccesso all'interno della cellula, l'eccedenza è traslocata nel vacuolo dove il metallo è memorizzato in molecole di ferritina8.
Ferro da stiro può essere misurata nei tessuti vegetali da diversi metodi, tra cui fiamma spettroscopia ad assorbimento atomico9 (FAAS) o saggi colorimetrici10, il primo è molto più precisa rispetto al secondo. FAAS è una tecnica estremamente precisa che consente di determinare la composizione elementare di un campione con l'emissione elettromagnetica dei singoli elementi. FAAS converte ioni metallici stati atomici di fiamma-riscaldamento del campione, che conduce agli ioni eccitazione e di emissione di una specifica lunghezza d'onda quando un determinato ione ritorna al suo stato fondamentale. Le emissioni da diversi ioni sono separate da un monocromatore e rilevate da un sensore di assorbimento11. FAAS quindi serve a quantificare direttamente le concentrazioni di ferro. Altre tecniche per la visualizzazione di ferro nei tessuti biologici sono, tuttavia, disponibili. Spettroscopia di massa del plasma ad accoppiamento induttivo (ICP-MS)12 è una tecnica molto precisa per la misurazione del ferro e altri oligoelementi, ma la mancanza di attrezzature, sia per FAAS e ICP-MS, è un problema comune. D'altra parte, misura del ferro di tiocianato colorimetria13 manca di precisione e non riesce a rilevare piccole variazioni tra campioni. 14,15,16,di colorazione blu di Prussia17 è un metodo indiretto basato sulla reazione di ferrocianuro ferrico di potassio (K4Fe(CN)6) con cationi di Fe, producendo un colore blu forte e viene utilizzato per la rilevazione qualitativa di ferro nelle sezioni istologiche dei tessuti animali e vegetali.
Ferro metallico (zero-valent) è rara nella litosfera. La forma ionica non complessato dominante di ferro nell'ambiente è principalmente dettata dalla quantità di ossigeno nei dintorni, con ferro ferroso, essendo relativamente più abbondante in ambienti anossici e ferro ferrico che predomina nei siti di aerobiche. Quest'ultima forma è anche dominante in ambienti estremamente acidi, anche se gli agenti causali di ossidazione del ferro ferroso spesso differiscono in un ambiente anossico e acide18. Quando il ferro viene solubilizzato in 4% HCl (pH 0) in un ambiente aerobico, la maggior parte del ferro diluito esiste come forma il ferrico (Fe3 +)19,20.
Le reazioni tra ioni Fe e K4Fe(CN)6 sono come segue:
Fe3 +: FeCl3 + K4Fe(CN)6 = KFe(III)Fe(II)(CN)6¯ + 3KCl
Fe2 +: 4 FeCl2 + 2K4Fe(CN)6 = Fe4(Fe(CN)6)2 + 8 KCl
Nello studio presente, abbiamo chiesto se la colorazione blu di Prussia può essere utile per misurare i livelli di ferro in soluzione.
Inizialmente, abbiamo verificato la correlazione tra la concentrazione di Fe in soluzione acquosa e colorazione blu di Prussia. La concentrazione di Fe (come FeCl2, FeCl3 o una miscela di 1:1 dei due) in soluzioni acquose è stata misurata sia di assorbanza (OD) di spettroscopia atomica dopo l'aggiunta del blu di Prussia. La figura 1 Mostra le curve di regressione lineare per misurazioni ottenute da ciascun metodo. Abbiamo concluso che il metodo prussiano blu può essere utilizzato per l'analisi quantitativa della concentrazione di ferro in soluzione.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57408/57408fig1.jpg"> Figura 1: regressione lineare tra concentrazione di Fe misurata FAAS e assorbanza della luce (OD, 715 nm) ottenuti con il metodo prussiano blu. I quadrati blu e linea rappresentano la soluzione di Fe2 + , i quadrati rossi e linea rappresentano la soluzione3 + Fe e i quadrati neri e linea rappresentano una miscela 1:1 tra Fe2 + e Fe3 +. Le regressioni seguenti sono state ottenute: [Fe2 +] = 3 + 123 x OD, r = 0,996, R2 = 0,989; [Fe3 +] = 1 + 292 x OD, r = 0,999, R2 = 0.997; e [Fe2 + 3 +] = 11 + 146 x OD, r = 0,983, R2 = 0.956. Il Fe2 + donatore era FeCl2 e il Fe3 + donatore era FeCl3. <a href="https://www.jove.com/files/ftp_upload/57408/57408fig1large.jpg" target="_blank">Clicca qui per visualizzare una versione più grande di questa figura.</a>
Per adattare il metodo colorimetrico blu di Prussia per l'analisi quantitativa di ferro dei tessuti vegetali, il contenuto di ferro delle ceneri di foglia di tabacco è stato misurato dalla spettroscopia di assorbimento atomico di fiamma e colorazione blu di Prussia. C'era buona correlazione tra i risultati di due tecniche.

<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

Ferro, uno dei più importanti micronutrienti negli organismi viventi, è coinvolto in processi di base, come la respirazione e la fotosintesi. Contenuto di ferro è piuttosto bassa in tutti gli organismi, pari a piante a circa 0,009% del peso secco. Ad oggi, uno dei metodi più precisi per misurare la concentrazione di ferro nei tessuti vegetali è fiamma spettroscopia di assorbimento atomico. Tuttavia, questo approccio è che richiede tempo e costoso e richiede attrezzature specifiche non comunemente trovate in laboratori di pianta. Pertanto, è necessario un metodo più semplice, ma preciso che può essere utilizzato regolarmente. Il metodo colorimetrico di blu di Prussia è regolarmente utilizzato per ferro qualitativo colorazione nelle sezioni istologiche di animali e vegetali. In questo studio, abbiamo adattato il metodo per la misurazione quantitativa del ferro nel tabacco lascia il blu di Prussia. Abbiamo validato la precisione di questo metodo utilizzando sia la spettroscopia atomica e blu di Prussia macchiatura per misurare il contenuto di ferro negli stessi campioni e trovato una regressione lineare (R2 = 0,988) fra le due procedure. Concludiamo che il metodo di blu di Prussia per la misurazione quantitativa del ferro nei tessuti vegetali è preciso, semplice e poco costoso. Tuttavia, la regressione lineare qui presentata non può essere appropriata per altre specie di piante, a causa di potenziali interazioni tra il campione e il reagente. Istituzione di una curva di regressione è quindi necessaria per diverse specie di piante.

Quando questo protocollo viene eseguito correttamente, si dovrebbe avere ottima correlazione tra i risultati ottenuti con i metodi di spettroscopia atomica e blu di Prussia. Pertanto, il metodo blu di Prussia può essere facilmente utilizzato per ottenere una misurazione accurata della concentrazione di ferro in campioni di piante, come risulta il seguente esperimento.
Piante di tabacco erano cresciute come descritto nel protoco...

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

Un metodo colorimetrico per la misurazione del ferro contenuto nelle piante

Vi presentiamo un protocollo semplice e affidabile per misurare il contenuto di ferro nei tessuti vegetali, utilizzando il metodo colorimetrico di blu di Prussia.

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.

Covalente legame degli anticorpi al cellulosa dischi di carta e le loro applicazioni a occhio nudo colorimetrici Immunoassays

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

Ricerca

Biochimica

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.

Un metodo per l&#39;RNA di misura<em&gt; N</em<sup&gt; 6</sup&gt; -methyladenosine modifiche di cellule e tessuti

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.

Un metodo per misurare il metabolismo in Ordinati sottopopolazioni di cellule Comunità complessi utilizzando Stable Isotope 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: Un metodo per acquisire e risolvere i complessi proteici nei campioni biologici

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.

La misurazione della legame proteico con F-actina mediante co-sedimentazione

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.

Determinazione del contenuto di glicogeno nei cianobatteri

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.

Caratterizzazione strutturale di Mannan polisaccaridi di parete cellulare nelle piante usando passo

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.

Un metodo semplice per High Throughput Screening chimico 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.

Determinazione spettrofotometrica del contenuto di Phycobiliprotein in 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.

Misurare e interpretare i tassi di consumo di ossigeno in tutto Vola testa segmenti

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