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

이 작품은 농업 (#16-16-0003)의 이스라엘 사역의 수석 과학자에서 교부 금에 의해 이스라엘 사역의 과학, 기술 및 Spaceand에 의해 지원 되었다.

1. 식물 재료와 성장 조건
<ol><li>씨앗 한 담배 (재배 삼 순) 씨 5 cm x 5 cm 냄비 당 표준 냄비 중간으로 가득합니다. 쟁반에 냄비를 놓습니다. 23 ° C의 일정 한 온도에서 긴 하루 조건 (16/8 h 명암) 성장 방에 식물을 성장 냄비에서 물 배수까지 수돗물 관개.</li>
	<li>50±5 일 후, 관개, 실험에 대 한 적당 한 농도 따라에서 Fe 치료를 시작 합니다. 예를 들어 우리는 철 농도 0에서 6 m m, 성 철 chelator (Fe EDDHA)에 ...

<ol>
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식물 조직에서 철 측정 관개 또는 다른 환경 조건의 효과 평가 하기 위한 매우 중요 하다. 여기, 우리는 다른 종의 식물와 조직에 쉽게 적응 될 수 있는 담배 잎에 Fe 내용 측정을 위한 간단 하 고 정확한 색도계 방법 설명.
색도계 방법에 대 한 조건 최적화, 우리는 철 용 해도를 낮은 pH 매체 (pH < 1.0) 사용 있습니다. 레코딩 프로세스는 모든 형태의 철을 해제 하 고 아무 오염 물?...

철 (Fe)은 모든 생명체에 중요 한 미 량 영양소 이다. 식물에는 필수 미 량 영양소1 호흡, 광합성과 엽록소 생 합성 등의 기본 프로세스에의 참여 때문 이다. 무료 철 이온의 높은 축적은 식물 세포 산화 스트레스를 일으키는 자유 래 디 칼의 방출에 지도 하는 반응 때문에 유해한. 식물 세포 내 철 항상성 유지, 이온 그들에 저장 하 고 ferritins, 철 항상성2 에 직접 관여 단백질 버팀대 및 모든 살아있는 유기 체에 있는 철의 주요한 저장소 구조 내에서 분리 된. 동시에 철 분 결핍 빈 혈 공장 철 영양실조 증가 필요에 따른 인간의 인구의 상당한 비율을 영향을 줍니다. 식물 ferritin의 독특한 특성으로 인해 ferritin 철 식품 농축 영양3의이 문제를 싸 우기 위하여 유망 전략을 제공 합니다.
철 이온은 주로에서 발견된 두 산화 상태, 즉 철 (divalent Fe2 + 또는 철 (II))과 제 2 철 (trivalent Fe3 + 또는 철 (III)) 형태. 철, 철 클러스터4, 등의 여러 다른 형태도 세포에서 발견 된다. Fe 철 산화물 세포와 자연스럽 게 폼 hematites (Fe2O3) 및 ferryhidrites로 저장 됩니다 ((Fe3 +)2O3•0.5 H2O)에서 생리 적 조건5. 이러한 반응, 특히 제 2 철 형태에서에서 형성 하는 수 산화물은 매우 낮은 용 해도 있다. 철 보존 따라서 솔루션의 pH에 의해 영향을 받는 이며 주로 pH 56위의 고체.
불 쌍 한 용 해도 및 Fe의 높은 반응성을 고려 하면 공장 조직 및 장기 들의 전송 적합 한 chelating 분자와 연결 되어야 합니다. 또한, 철과 제 2 철 양식1 사이 그것의 산화 상태를 제어 해야 합니다. 잎, 내는 철의 약 80% 한다, 엽록소와 다른 heme 분자의 생 합성에서 전자 교통 시스템에서의 필수적인 역할 때문에 광합성 세포에서 발견 되 고 Fe S의 형성에서 클러스터7. 셀 내에서 과잉 철 분의 경우 흑자 금속 ferritin 분자8에 저장 하는 공포에 translocated입니다.
철은 여러 방법을 비롯 하 여 불꽃 원자 흡수 분광학9 (FAAS) 또는 색도계 분석 실험10, 전 후자 보다 훨씬 더 정확한 되 고 식물 조직에서 측정할 수 있습니다. FAAS 있는 개별 요소의 전자기 방출 기초 샘플의 원소 구성을 결정 하는 매우 정확한 기술입니다. FAAS 변환 합니다 금속 이온 원자 상태 샘플의 화 염 열에 의해 주어진된 이온 접지 상태로 반환 될 때 이온 여기 및 특정 파장의 방출으로 이어지는. 다른 이온에서 방출 한 단색으로 구분 되며 흡수 센서11에 의해 감지. FAAS 따라서 직접 철 농도 척도를 제공 합니다. 그러나 생물 조직에 철을 시각화에 대 한 다른 기술을,, 사용할 수 있습니다. 유도 결합 플라즈마-질량 분광학 (ICP-MS)12 철 및 다른 성분 측정을 위한 매우 정확한 방법 이지만 장비, FAAS와 ICP-MS, 모두의 부족은 일반적인 문제 이다. 다른 한편으로, 안산 기능성13 철 측정 정밀도 결여 하 고 샘플 사이의 작은 변화를 감지 하 실패 합니다. 프러시안 파란 얼룩14,,1516,17 Fe 양이온, 생산 되는 칼륨 제 2 철 ferrocyanide (K4Fe(CN)6)의 반응에 따라 간접 방법입니다는 강한 블루 색상, 질적 철 검출 동물과 식물 조직의 조직학 섹션에 사용 됩니다.
철 금속 (0-발렌타인)는 암석에 드문 경우입니다. 환경에서 철의 지배적인 비 complexed 이온 형태로 주로 에어로빅 사이트에서 우세한 제 2 철 철 철 철 무산 소 환경에서 상대적으로 더 풍부한 되 고와 주변에 있는 산소의 양에 의해 결정 됩니다. 철 철 산화의 원인이 되는 에이전트 자주 무산 소 및 산 성 주변18에 차이가 있지만이 후자의 형태는 또한 극단적으로 산 성 환경에서 지배적 이다. 때 철 분 4%에서 solubilized 이다 호 기성 환경에서 HCl (pH 0), 희석된 철의 존재는 제 2 철 (Fe3 +)19,20양식.
철 이온과 K4Fe(CN)6 사이의 반응을 아래와 같습니다.
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
현재 연구에서 우리는 프러시안 파란 얼룩 솔루션에서 철 수준을 측정 하는 데 유용 수 있습니다 여부를 물었다.
처음에, 수성 해결책에서 Fe의 농도 및 프러시안 파란 얼룩의 상관관계를 확인합니다. 수성 해결책에서 (FeCl2, FeCl3 또는 2의 1:1 혼합물)로 철 농도 프러시안 블루의 추가 후 원자 분광학와 흡 광도 (OD)을 측정 했다. 그림 1 측정 각 방법으로 얻은 선형 회귀 곡선을 보여준다. 우리는 프러시안 블루 메서드 솔루션에서 철 분 농도의 정량 분석을 위해 사용 될 수 있다는 결론을 내렸다.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57408/57408fig1.jpg"> 그림 1: Fe 농도 사이의 선형 재발 FAAS와 빛 흡 광도 측정 (OD, 715 nm) 프러시안 블루 메서드에서 얻은. 파란색 사각형 및 선 Fe2 + 솔루션, 빨간 사각형 및 선 Fe3 + 솔루션 및 검은 사각형을 나타냅니다 나타내고 선 사이 Fe2 + Fe3 +1:1 혼합물을 나타냅니다. 다음 재발 얻어 졌다: [Fe2 +] 3 + 123 x OD = r = 0.996, R2 0.989; = [Fe3 +] 1 + 292 x OD = r = 0.999, R2 = 0.997; 그리고 [Fe2 + 3 +] 11 + 146 x OD = r = 0.983, R2 0.956 =. Fe2 + 기증자 FeCl2 이었고 Fe3 + 기증자 FeCl3했다. <a href="https://www.jove.com/files/ftp_upload/57408/57408fig1large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭 하십시오.</a>
식물 조직의 철 정량 분석에 대 한 색도계 프러시안 블루 방법에 맞게, 담배 잎 재 철 콘텐츠 불꽃 흡수 원자 분광학 및 프러시안 파란 얼룩에 의해 측정 되었다. 두 가지 방법에 의해에서 결과 사이 좋은 상관 관계가 있었습니다.

<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

철, 살아있는 유기 체에서 가장 중요 한 영양소 중 하나는 호흡과 광합성 등의 기본 프로세스에 포함 된다. 철 분 모든 생물, 식물에 건조 중량의 약 0.009%에 이른다에 오히려 낮습니다. 날짜 하려면, 화 염 흡수 원자 분광학은 식물 조직에서 철 분 농도 측정 하기 위한 가장 정확한 방법 중 하나. 그러나,이 접근은 어렵고 비싼 이며 특정 장비 공장 실험실에서 주로 발견. 따라서, 정기적으로 사용 될 수 있는 간단한 아직 정확한 방법 필요 합니다. 색도계 프러시안 블루 메서드는 정기적으로 동물과 식물 조직학 섹션에 얼룩 질적 철에 사용 됩니다. 이 연구에서 우리는 담배에 있는 철의 양적 측정 방법 나뭇잎 프러시안 블루 적응. 우리는 원자 분광학과 같은 샘플에 철 콘텐츠를 측정 하 여 선형 회귀 발견 얼룩 프러시안 블루를 사용 하 여이 방법의 정확도 확인 (R2 = 0.988) 두 절차 사이. 우리는 프러시안 블루 식물 조직에서 양적 철 측정 방법은, 간단, 정확 하 고 저렴 한 결론. 그러나, 여기에 제시 된 선형 회귀 다른 식물 종, 샘플 및 시 사이 잠재적인 상호 작용 때문에 적합 하지 않을 수 있습니다. 따라서 회귀 곡선의 다른 식물 종의 필요 합니다.

이 프로토콜은 제대로 실시는, 하나 우수한 상관관계 프러시안 블루와 원자 분광학 방법으로 얻은 결과 얻어야 한다. 따라서, 프러시안 블루 메서드를 사용할 수 있습니다 쉽게 식물 샘플에서 철 분 농도의 정확한 측정을 얻기 위해 다음과 같은 실험에 반영.
담배 식물은 프로토콜에 설명 된 대로 성장 하 고 다른 철 농도 포함 ...

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

식물에 철 분 함량을 측정 하는 색도계 방법

선물이 색도계 프러시안 블루 메서드를 사용 하 여 식물 조직에 철 분 함량을 측정 하는 간단 하 고 신뢰할 수 있는 프로토콜.

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.

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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.

셀룰로오스 종이 디스크와 벌거 벗은 눈 색채 면역 측정법에 그들의 응용 프로그램에 항체의 결합 공유

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

연구

생화학

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.

RNA 측정 방법<em&gt; N</em<sup&gt; 6</sup세포와 조직에서&gt; -methyladenosine 수정

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.

안정 동위 원소 추적을 사용하여 복잡한 세포 공동체의 정렬 된 부분 집단에서 대사를 측정하는 방법

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.

다량 체-PAGE : 생물학적 샘플에서 캡처하는 방법 및 해결 단백질 단지

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.

공동 침강에 의한 F-actin에 대한 단백질 결합 측정

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.

시아 노 박테리아의 글리코겐 함량 측정

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.

속도 사용 하 여 식물에 Mannan 세포 벽 다 당 류 구조 특성

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.

꼬마 선 충 에서 높은 처리량 화학 심사 하는 간단한 방법

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.

Spectrophotometric Cyanobacterium Synechocystis 에서 Phycobiliprotein 콘텐츠 결정

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.

측정 및 해석의 전체 산소 소비 속도 비행 머리 세그먼트

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