Mali destek için Çin Bilim ve Teknoloji Bakanlığı&#39;na (2016YDF0600803) teşekkür ediyoruz.

1. Numune Hazırlama <ol><li> Bitki örneğinden küçük bir doku bloğu (yaklaşık 3 mm x 3 mm x 5 mm) kesin ( örn. Kavak kökü). </li><li> Dokuyu 30 dakika kaynar deiyonize suya daldırın. Oda sıcaklığında (RT) 30 dakika boyunca hemen deiyonize suya aktarın. Doku, kabın altına sızana kadar bu adımı tekrarlayın; dokudaki havanın alınmış olduğunu ve dokunun yumuşadığını belirtin. Not: Bu adımdan önce altına batan numuneler için, genellikle bu döngüyü 3-5 kez tekrarlayın. </li><li> Deiyonize suyun içindeki 20, 50, 70, 90% (h / h) miktarlardaki polietilen glikol (PEG) ve saf PEG&#39;yi hazırlayın ve solüsyonları 65 ° C&#39;de tutun. </li><li> Suyu, yerinden çıkartmak ve PEG&#39;nin sızmasına izin vermek için, dokuyu bir dizi dereceli PEG banyosunda inkübe edin. Not: Genellikle, molekül ağırlığı 2,000 olan (PEG2000) PEG, gömme için kullanılır. <ol><li> Doku g ile işleyin(A) 1 saat boyunca% 20 PEG, (b) 1.5 saat boyunca% 50 PEG, (c) 2 saat boyunca% 70 PEG, (d) 2 saat boyunca% 90 PEG, ve (E)% 10 PEG ile 10 saat. </li></ol></li><li> Kaseti 65 ° C&#39;de fırında önceden ısıtın. Bloğu içeren PEG&#39;yi kasete dökün ve daha sonra önceden ısıtılmış cımbız veya iğne kullanarak doku bloğunu istenen bir yere yerleştirin. </li><li> Kaseti yavaşça soğutun ve doku kullanıma kadar oda sıcaklığında saklayın. </li><li> Keskin bir tıraş bıçağı kullanarak küçük bir blok (yaklaşık 1 cm x 1 cm x 2 cm) içine hedef doku içeren PEG bloğu parçalayın ve mikrotom üzerine monte edin. </li><li> PEG bloğundan ince kesitleri kesin (tipik olarak 3-10 μm). </li><li> PEG&#39;yi dokudan çıkartmak için, bölmeyi deiyonize su ile bir saat camında 10 kez durulayın. </li><li> Ekstraktları çıkarmak için bölümleri toluen / etanol (2: 1, v / v) ile 6 saat bekletin. 65 mL deiyonize su, 0.5 mL asetik asit ve 0.6 g sodyum klorür karıştırılarak reaksiyon sıvısı hazırlanır.Bir kapta ritüel edin. 5 mL şişeye bir bölüm ve 3 mL reaksiyon sıvısı ekleyin. Şişenin üst kısmını vidalayın. </li><li> Dokunun lignini çıkarmak için şişeyi 75 ° C&#39;de 2 saat boyunca su banyosunda ısıtın. Parçayı deiyonize su ile 10 kez saat camında yıkayın. </li><li> İşlenmemiş / tasfiye edilmemiş bölümü cam mikroskop lamı üzerine aktarın. Fırçaları veya iğneleri kullanarak bölümü dikkatle açın. Ekstra deiyonize suyu kağıttan kağıtla çıkarın. </li><li> Kesiti D 2 O içine sokun. Numuneyi bir cam kapak folyosuyla örtün. D2O&#39;nun buharlaşmasını önlemek için kapak kağıdını oje ile mühürle. </li></ol> 2. Spektral Edinme <ol><li> Konfokal Raman mikroskopunun cihaz çalıştırma yazılımını açın. Lazeri açın (dalga boyu = 532 nm), 100X mikroskop objektifli kristal silisyumun yüzeyine odaklayın ve cihazı kalibre etmek için kalibrasyon düğmesine tıklayın. </li><li> Aleti değiştirOptik mikroskop moduna geçirin ve mikroskop lambasını açın. Mikroskop lamı, objektife bakacak şekilde kapak kaymasıyla birlikte sahneye monte edin. </li><li> 20X mikroskop objektifle örneği inceleyin ve ilgi alanını bulun. Lamel içine daldırma yağı uygulayın ve daldırmalı mikroskop objektif (60X, sayısal diyafram NA = 1.35) geçiş yapın. Numunenin yüzeyine odaklanın. </li><li> Cihazı, Raman test moduna getirin ve mikroskop lambasını kapatın. </li><li> Dikdörtgen bir araç kullanarak bir haritalama alanı seçin. Elde edilen spektrumların sayısını belirlemek için adım boyutunu değiştirin. Not: Adım boyutunun farkında olun (genellikle objektifin sayısal diyaframı ile hesaplanan spot çapından daha büyük, teorik olarak 1.22λ / NA). Bunun altındaki boyutlar aşırı örneklemeye neden olur. </li><li> Uygun bir edinim zamanında (genellikle &lt;8 saat) en iyi sinyal-gürültü oranı (SNR) ve spektral kaliteyi elde etmek için optimum spektral parametreleri ayarlayın, depeNumunenin uygunluğuna değinerek. Genellikle, görüntüleme parametrelerini cihaz yazılımında aşağıdaki gibi girin: lazer (532 nm), filtre (100%), delik (300), yarık (100), spektrometre (1,840 cm -1 ), oluklar (1200 t), objektif 60X yağ) ve edinme süresi (2 s). </li><li> Veri işleme öncesinde spektral verileri saklayın ve evrensel bir biçime ( ör. TXT dosyaları) dönüştürün. </li></ol> 3. Veri Analizi <ol><li> Spektral verileri (TXT dosyaları) veri analizi yazılımına yükleyin ( örn., Matlab). SNR&#39;yi iyileştirmek için veri kümesindeki bir gürültü azaltma tekniği uygulayın ( örneğin, Savitzsky-Golay algoritması veya dalgacık algoritması) </li><li> Lignin ve polisakarit görüntüleri üretmek için işlem görmemiş numuneler kullanın. Lignin görüntülemede, aromatik halka simetrik gerilme titreşiminden dolayı 1,600 cm -1 civarında spektral zirveyi göz önünde bulundurun. Polisakkarit görüntüleme (selüloz ve hemiselüloz dahil) için, spektral zirveyi aCH ve CH 2 gerilmelerinden dolayı 2,889 cm -1 yuvarlaktır. </li><li> Selüloz ve hemiselüloz görüntüleri oluşturmak için lekeleyici örnekleri kullanın. Elde edilen spektrumlarda selüloz ve hemiselülozun spektrumlarını ayırt etmek ve dağılımlarını görüntülemek için kendinden modelleme eğrisi çözünürlüğü (SMCR) uygulayın. </li><li> Farklı hücre duvarı katmanlarından Raman spektrumlarını ayırt etmek için elde edilen verilere temel bileşen analizi (PCA) ve kümeleme analizi uygulayın. </li></ol>

<ol>
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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+2.">Chapter 2.</a> Ionic Liquids in the Biorefinery Concept: Challenges and Perspectives. , 38-64 (2016).</li><li>Sun, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding+tissue+specific+compositions+of+bioenergy+feedstocks+Through+hyperspectral+Raman+imaging.">Understanding tissue specific compositions of bioenergy feedstocks Through hyperspectral Raman imaging.</a> Bio. 108 (2), 286-295 (2009).</li><li>Tolstik, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Classification+and+prediction+of+HCC+tissues+by+Raman+imaging+with+identification+of+fatty+acids+as+potential+lipid+biomarkers.">Classification and prediction of HCC tissues by Raman imaging with identification of fatty acids as potential lipid biomarkers.</a> J. Cancer. Res. Clin. Oncol. 141 (3), 407-418 (2015).</li><li>Schrader, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Infrared and Raman spectroscopy: methods and applications. , (2008).</li><li>Gierlinger, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+of+plant+cell+walls+by+confocal+Raman+microscopy.">Imaging of plant cell walls by confocal Raman microscopy.</a> Nat. Protoc. 7 (9), 1694-1708 (2012).</li><li>Luca, A. C. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Online+fluorescence+suppression+in+modulated+Raman+spectroscopy.">Online fluorescence suppression in modulated Raman spectroscopy.</a> Anal. 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Raman Spectrosc. 40 (12), 1720-1725 (2009).</li><li>Lawton, W. H., Sylvestre, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self+modeling+curve+resolution.">Self modeling curve resolution.</a> Technometrics. 13, 617-633 (1971).</li><li>Zhang, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Method+for+automatically+identifying+spectra+of+different+wood+cell+wall+layers+in+Raman+imaging+data+set.">Method for automatically identifying spectra of different wood cell wall layers in Raman imaging data set.</a> Anal. Chem. 87 (2), 1344-1350 (2015).</li><li>Kudelski, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analytical+application+of+Raman+spectroscopy.">Analytical application of Raman spectroscopy.</a> Talanta. 76 (1), 1-8 (2008).</li></ol>

Yazarların açıklayacak bir şeyi yok.

Bitki hücre duvarı, hücre köşesi (CC), ikincil duvar (SW, S1, S2 ve S3 katmanları) ve bileşik orta lamel (CML, orta lamel artı birincil birincil Duvar), bu da numunenin hazırlanması sırasında düz bir yüzey elde edilmesini zorlaştırmaktadır. Bu nedenle, ahşaptan daha karmaşık bir yapıya sahip bitki örnekleri, özellikle çim, çoğunlukla ince kesit vermeye izin vermek için katılaştırılmaya ihtiyaç duyar. PEG, suda çözünür olduğu için kesme ve Raman araştırması için ideal bir sert ma...

Plant biomass is the most abundant renewable resource on Earth; is mainly composed of lignin, cellulose, and hemicellulose; and is considered an attractive source of bioenergy and bio-based chemicals1. Unfortunately, it can resist degradation and confer hydrolytic stability or structural robustness to the plant cell wall. Such resistance is attributable to the accessible surface area, biomass particle size, degree of polymerization, cellulose crystallinity, and protective lignin2. A comprehensive understanding of the structural and chemical nature of the plant cell wall is thus significant from the viewpoint of plant biology and chemistry, as well as from that of commercial utilization. Commonly used wet chemistry analyses, such as chromatography, mass spectrometry, and nuclear magnetic resonance spectroscopy, only provide average compositional data of the measured sample. Furthermore, these methods are invasive and destroy the original structure of the plant tissue3. 
The Raman imaging technique is a powerful tool for the nondestructive visualization of spatially resolved chemical information4. It uses a laser light to cause inelastic scattering with a photon and relies on changes in polarizability arising from the molecular vibrations. In this case, water causes weak Raman scattering, which makes this approach suitable for in situ investigations of biological samples5. The application of the Raman imaging technique to the plant cell wall can elucidate the structure and composition of plant cell walls in their native state, with the resolution on the scale of the single cell and even of the cell wall layers6. A typical Raman imaging analysis of a plant cell wall generally consists of three steps: 1) sample preparation, 2) spectral acquisition, and 3) data processing. 
Although one of the major advantages of Raman imaging is the ability to achieve label-free and non-destructive spectra with minimal sample preparation, physical sample sectioning is still necessary to expose the surface of interest. This process should be performed carefully to obtain a flat surface, since the technique depends on maintaining optical focus7. Spectral acquisition requires a balance between image quality and extensive acquisition times8. Data processing aims to effectively extract the chemical information from the image data, especially for the components with similar chemical structures, such as cellulose and hemicellulose. Due to the strong spectral overlap, the exact spectra are difficult to discern. In this case, multivariate analysis is a straightforward approach to effectively uncover the hiding structural and chemical information9. This work presents a general protocol describing the use of Raman imaging to visualize the main components in plant cell walls, including lignin, cellulose, and hemicellulose.

<table border="0" cellpadding="0" cellspacing="0" width="597">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:199px;">Microtome</td>
			<td style="width:136px;">Thermo Scientific</td>
			<td style="width:199px;">Microm HM430</td>
			<td style="width:64px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Confocal Raman microscope</td>
			<td>Horiba Jobin Yvon</td>
			<td>Xplora</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Oven</td>
			<td>Shanghai ZHICHENG</td>
			<td>ZXFD-A5040</td>
			<td></td>
		</tr>
	</tbody>
</table>

combining raman imaging and multivariate analysis to visualize lignin, cellulose, and hemicellulose in the plant cell wall

Raman görüntüsünün bitki biyokütle uygulamasına uygulanması, sulu çözeltiler üzerinde mekansal ve kompozisyonsal bilgi sunabileceği için artmaktadır. Analiz, genellikle kapsamlı numune hazırlığı gerektirmez; Yapısal ve kimyasal bilgiler etiketsiz olarak elde edilebilir. Bununla birlikte, her Raman görüntüsü binlerce spektrum içermektedir; Gizli bilgileri ayıklarken, özellikle de benzer kimyasal yapıya sahip bileşenler için güçlükler doğar. Bu çalışma, bu sorunu çözmek için çok değişkenli bir analizi ortaya koymaktadır. Protokol, bitki hücre duvarı içerisindeki lignin, selüloz ve hemiselüloz dahil olmak üzere ana bileşenleri görselleştirmek için genel bir yöntem oluşturmaktadır. Bu protokolde, numune hazırlama, spektral edinme ve veri işleme prosedürleri anlatılmıştır. Numune hazırlama ve veri analizinde operatör becerisine oldukça bağımlıdır. Bu yaklaşımı kullanarak, uzman olmayan bir kullanıcı tarafından bir Raman araştırması yapılabilir.H kalite verisi ve bitki hücre duvar analizi için anlamlı sonuçlar.

Şekil 1 , bir bitki hücre duvarının Raman görüntüsü için tipik bir mikro-Raman sistemine genel bir bakış sunmaktadır. Örnek olarak, kavak ( Populus nigra L.) orijinal Raman spektrumlarında belirgin taban çizgileri ve sivri uçlar bulunur ( Şekil 2a ). Raman görüntüleme veri seti (APRI) için otomatik ön işleme yöntemini yaptıktan sonra, bu iki spektral kirletici başarıyla kaldırılır ( <strong...

Watch this Scientific Journal Video about Combining Raman Imaging and Multivariate Analysis to Visualize Lignin, Cellulose, and Hemicellulose in the Plant Cell Wall at JoVE.com

Combining Raman Imaging and Multivariate Analysis to Visualize Lignin, Cellulose, and Hemicellulose in the Plant Cell Wall

Bu protokol, bitki hücre duvarlarındaki lignin, selüloz ve hemiselülozun Raman görüntüleme ve çok değişkenli analizi kullanılarak görselleştirilmesi için genel bir yöntem sunmayı amaçlamaktadır.

Bitki Hücresi Duvarında Lignin, Selüloz ve Hemiselülozu Görselleştirmek için Raman Görüntüleme ve Çok Değişkenli Analizin Birleştirilmesi

The application of Raman imaging to plant biomass is increasing because it can offer spatial and compositional information on aqueous solutions. The ...

combining-raman-imaging-multivariate-analysis-to-visualize-lignin

Universidad San Sebastian

Research

JoVE Journal

Biochemistry

11.8K Görüntüleme.  Beijing Forestry University.  Bu protokol, bitki hücre duvarlarındaki lignin, selüloz ve hemiselülozun Raman görüntüleme ve çok değişkenli analizi kullanılarak görselleştirilmesi için genel bir yöntem sunmayı amaçlamaktadır. 

Video: Combining Raman Imaging and Multivariate Analysis to Visualize Lignin, Cellulose, and Hemicellulose in the Plant Cell Wall

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.

Siyanobakterlerde Glikojen İçeriğinin Belirlenmesi

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

Biyokimya

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.

Yapısal Mannan hücre duvarı polisakkaritler hızı kullanarak tesislerinde karakterizasyonu

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

Isolation and Respiratory Measurements of Mitochondria from Arabidopsis thaliana

Mitochondria are essential organelles involved in numerous metabolic pathways in plants, most notably the production of adenosine triphosphate (ATP) from the oxidation of reduced compounds such as nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FADH2). The complete annotation of the Arabidopsis thaliana genome has established it as the most widely used plant model system, and thus the need to purify mitochondria from a variety of organs (leaf, root, or flower) is necessary to fully utilize the tools that are now available for Arabidopsis to study mitochondrial biology. Mitochondria are isolated by homogenization of the tissue using a variety of approaches, followed by a series of differential centrifugation steps producing a crude mitochondrial pellet that is further purified using continuous colloidal density gradient centrifugation. The colloidal density material is subsequently removed by multiple centrifugation steps. Starting from 100 g of fresh leaf tissue, 2 - 3 mg of mitochondria can be routinely obtained. Respiratory experiments on these mitochondria display typical rates of 100 - 250 nmol O2 min-1 mg total mitochondrial protein-1 (NADH-dependent rate) with the ability to use various substrates and inhibitors to determine which substrates are being oxidized and the capacity of the alternative and cytochrome terminal oxidases. This protocol describes an isolation method of mitochondria from Arabidopsis thaliana leaves using continuous colloidal density gradients and an efficient respiratory measurements of purified plant mitochondria.

Isolation and Respiratory Measurements of Mitochondria from Arabidopsis thaliana

As mitochondria are only a small percentage of the plant cell, they need to be purified for a range of studies. Mitochondria can be isolated from a variety of plant organs by homogenization, followed by differential and density gradient centrifugation to obtain a highly purified mitochondrial fraction.

Yalıtım ve solunum ölçümleri mitokondri Arabidopsis thaliana üzerinden

Mitochondria are essential organelles involved in numerous metabolic pathways in plants, most notably the production of adenosine triphosphate (ATP) from ...

2 in 1: One-step Affinity Purification for the Parallel Analysis of Protein-Protein and Protein-Metabolite Complexes

Cellular processes are regulated by interactions between biological molecules such as proteins, metabolites, and nucleic acids. While the investigation of protein-protein interactions (PPI) is no novelty, experimental approaches aiming to characterize endogenous protein-metabolite interactions (PMI) constitute a rather recent development. Herein, we present a protocol that allows simultaneous characterization of the PPI and PMI of a protein of choice, referred to as bait. Our protocol was optimized for Arabidopsis cell cultures and combines affinity purification (AP) with mass spectrometry (MS)-based protein and metabolite detection. In short, transgenic Arabidopsis lines, expressing bait protein fused to an affinity tag, are first lysed to obtain a native cellular extract. Anti-tag antibodies are used to pull down protein and metabolite partners of the bait protein. The affinity-purified complexes are extracted using a one-step methyl tert-butyl ether (MTBE)/methanol/water method. Whilst metabolites separate into either the polar or the hydrophobic phase, proteins can be found in the pellet. Both metabolites and proteins are then analyzed by ultra-performance liquid chromatography-mass spectrometry (UPLC-MS or UPLC-MS/MS). Empty-vector (EV) control lines are used to exclude false positives. The major advantage of our protocol is that it enables identification of protein and metabolite partners of a target protein in parallel in near-physiological conditions (cellular lysate). The presented method is straightforward, fast, and can be easily adapted to biological systems other than plant cell cultures.

Protein-protein and protein-metabolite interactions are crucial for all cellular functions. Herein, we describe a protocol that allows parallel analysis of these interactions with a protein of choice. Our protocol was optimized for plant cell cultures and combines affinity purification with mass spectrometry-based protein and metabolite detection.

2 in 1: tek adımlı benzeşme arıtma paralel analizi için Protein-Protein ve Protein-metaboliti kompleksleri

Cellular processes are regulated by interactions between biological molecules such as proteins, metabolites, and nucleic acids. While the investigation of ...

Using In Vitro Fluorescence Resonance Energy Transfer to Study the Dynamics Of Protein Complexes at a Millisecond Time Scale

Proteins are the primary operators of biological systems, and they usually interact with other macro- or small molecules to carry out their biological functions. Such interactions can be highly dynamic, meaning the interacting subunits are constantly associated and dissociated at certain rates. While measuring the binding affinity using techniques such as quantitative pull-down reveals the strength of the interaction, studying the binding kinetics provides insights on how fast the interaction occurs and how long each complex can exist. Furthermore, measuring the kinetics of an interaction in the presence of an additional factor, such as a protein exchange factor or a drug, helps reveal the mechanism by which the interaction is regulated by the other factor, providing important knowledge for the advancement of biological and medical research. Here, we describe a protocol for measuring the binding kinetics of a protein complex that has a high intrinsic association rate and can be dissociated quickly by another protein. The method uses fluorescence resonance energy transfer to report the formation of the protein complex in vitro, and it enables monitoring the fast association and dissociation of the complex in real time on a stopped-flow fluorimeter. Using this assay, the association and dissociation rate constants of the protein complex are quantified.

Protein-protein interactions are critical for biological systems, and studies of the binding kinetics provide insights into the dynamics and function of protein complexes. We describe a method that quantifies the kinetic parameters of a protein complex using fluorescence resonance energy transfer and the stopped-flow technique. &#160;

Protein kompleksleri milisaniyelik zaman ölçekte dinamikleri çalışmaya vitro Floresans rezonans enerji transferi içinde kullanma

Proteins are the primary operators of biological systems, and they usually interact with other macro- or small molecules to carry out their biological ...

In Vivo Calcium Imaging in C. elegans Body Wall Muscles

The model organism C. elegans provides an excellent system to perform in vivo calcium imaging. Its transparent body and genetic manipulability allow for the targeted expression of genetically encoded calcium sensors. This protocol outlines the use of these sensors for the in vivo imaging of calcium dynamics in targeted cells, specifically the body wall muscles of the worms. By utilizing the co-expression of presynaptic channelrhodopsin, stimulation of acetylcholine release from excitatory motor neurons can be induced using blue light pulses, resulting in muscle depolarization and reproducible changes in cytoplasmic calcium levels. Two worm immobilization techniques are discussed with varying levels of difficulty. Comparison of these techniques demonstrates that both approaches preserve the physiology of the neuromuscular junction and allow for the reproducible quantification of calcium transients. By pairing optogenetics and functional calcium imaging, changes in postsynaptic calcium handling and homeostasis can be evaluated in a variety of mutant backgrounds. Data presented validates both immobilization techniques and specifically examines the roles of the C. elegans sarco(endo)plasmic reticular calcium ATPase and the calcium-activated BK potassium channel in the body wall muscle calcium regulation.

In Vivo Calcium Imaging in C. elegans Body Wall Muscles

This method provides a way to couple optogenetics and genetically encoded calcium sensors to image baseline cytosolic calcium levels and changes in evoked calcium transients in the body wall muscles of the model organism C. elegans.

In Vivo Kalsiyum Görüntüleme C. elegans Vücut Duvar Kasları

The model organism C. elegans provides an excellent system to perform in vivo calcium imaging. Its transparent body and genetic manipulability allow for ...

Study on the Metabolism of Six Systemic Insecticides in a Newly Established Cell Suspension Culture Derived from Tea (Camellia Sinensis L.) Leaves

A platform for studying insecticide metabolism using in vitro tissues of tea plant was developed. Leaves from sterile tea plantlets were induced to form loose callus on Murashige and Skoog (MS) basal media with the plant hormones 2,4-dichlorophenoxyacetic acid (2,4-D, 1.0 mg L-1) and kinetin (KT, 0.1 mg L-1). Callus formed after 3 or 4 rounds of subculturing, each lasting 28 days. Loose callus (about 3 g) was then inoculated into B5 liquid media containing the same plant hormones and was cultured in a shaking incubator (120 rpm) in the dark at 25 &#177; 1 &#176;C. After 3&#8722;4 subcultures, a cell suspension derived from tea leaf was established at a subculture ratio ranging between 1:1 and 1:2 (suspension mother liquid: fresh medium). Using this platform, six insecticides (5 &#181;g mL-1 each thiamethoxam, imidacloprid, acetamiprid, imidaclothiz, dimethoate, and omethoate) were added into the tea leaf-derived cell suspension culture. The metabolism of the insecticides was tracked using liquid chromatography and gas chromatography. To validate the usefulness of the tea cell suspension culture, the metabolites of thiamethoxan and dimethoate present in treated cell cultures and intact plants were compared using mass spectrometry. In treated tea cell cultures, seven metabolites of thiamethoxan and two metabolites of dimethoate were found, while in treated intact plants, only two metabolites of thiamethoxam and one of dimethoate were found. The use of a cell suspension simplified the metabolic analysis compared to the use of intact tea plants, especially for a difficult matrix such as tea.

Study on the Metabolism of Six Systemic Insecticides in a Newly Established Cell Suspension Culture Derived from Tea Camellia Sinensis L. Leaves

This work presents a protocol for establishing a cell suspension culture derived from tea (Camellia sinensis L.) leaves that can be used to study the metabolism of external compounds that can be taken up by the whole plant, such as insecticides.

Çay (Camellia sinensis L.) türetilmiştir yeni kurulan hücre süspansiyon kültürü altı sistemik Böcek Ilacı metabolizması üzerinde çalışma Yaprak -ları

A platform for studying insecticide metabolism using in vitro tissues of tea plant was developed. Leaves from sterile tea plantlets were induced to form ...

Measuring Interactions between Fluorescent Probes and Lignin in Plant Sections by sFLIM Based on Native Autofluorescence

In lignocellulosic biomass (LB), the activity of enzymes is limited by the appearance of non-specific interactions with lignin during the hydrolysis process, which maintains enzymes far from their substrate. Characterization of these complex interactions is thus a challenge in complex substrates such as LB. The method here measures molecular interactions between fluorophore-tagged molecules and native autofluorescent lignin, to be revealed by F&#246;rster resonance energy transfer (FRET). Contrary to FRET measurements in living cells using two exogenous fluorophores, FRET measurements in plants using lignin is not trivial due to its complex autofluorescence. We have developed an original acquisition and analysis pipeline with correlated observation of two complementary properties of fluorescence: fluorescence emission and lifetime. sFLIM (spectral and fluorescent lifetime imaging microscopy) provides the quantification of these interactions with high sensitivity, revealing different interaction levels between biomolecules and lignin.

This protocol describes an original setup combining spectral and fluorescence lifetime measurements to evaluate F&#246;rster resonance energy transfer (FRET) between rhodamine-based fluorescent probes and lignin polymer directly in thick plant sections.

Floresan Problar ve Lignin Bitki Kesitlerinde Doğal Otofloresan Dayalı SFLIM Ile Etkileşimlerinin Ölçülmesi

In lignocellulosic biomass (LB), the activity of enzymes is limited by the appearance of non-specific interactions with lignin during the hydrolysis ...

An Integrated Raman Spectroscopy and Mass Spectrometry Platform to Study Single-Cell Drug Uptake, Metabolism, and Effects

Cells are known to be inherently heterogeneous in their responses to drugs. Therefore, it is essential that single-cell heterogeneity is accounted for in drug discovery studies. This can be achieved by accurately measuring the plethora of cellular interactions between a cell and drug at the single-cell level (i.e., drug uptake, metabolism, and effect). This paper describes a single-cell Raman spectroscopy and mass spectrometry (MS) platform to monitor metabolic changes of cells in response to drugs. Using this platform, metabolic changes in response to the drug can be measured by Raman spectroscopy, while the drug and its metabolite can be quantified using mass spectrometry in the same cell. The results suggest that it is possible to access information about drug uptake, metabolism, and response at a single-cell level.

This protocol presents an integrated Raman spectroscopy-mass spectrometry (MS) platform that is capable of achieving single-cell resolution. Raman spectroscopy can be used to study cellular response to drugs, while MS can be used for targeted and quantitative analysis of drug uptake and metabolism.

Tek Hücreli İlaç Alımı, Metabolizması ve Etkileri Üzerine Entegre Raman Spektroskopisi ve Kütle Spektrometresi Platformu

Cells are known to be inherently heterogeneous in their responses to drugs. Therefore, it is essential that single-cell heterogeneity is accounted for in ...

RNA Blot Analysis for the Detection and Quantification of Plant MicroRNAs

MicroRNAs (miRNAs) are a class of endogenously expressed non-coding, ~21 nt small RNAs involved in the regulation of gene expression in both plants and animals. Most miRNAs act as negative switches of gene expression targeting key genes. In plants, primary miRNAs (pri-miRNAs) transcripts are generated by RNA polymerase II, and they form varying lengths of stable stem-loop structures called pre-miRNAs. An endonuclease, Dicer-like1, processes the pre-miRNAs into miRNA-miRNA* duplexes. One of the strands from miRNA-miRNA* duplex is selected and loaded onto Argonaute 1 protein or its homologs to mediate the cleavage of target mRNAs. Although miRNAs are key signaling molecules, their detection is often carried out by less than optimal PCR-based methods instead of a sensitive northern blot analysis. We describe a simple, reliable, and extremely sensitive northern method that is ideal for the quantification of miRNA levels with very high sensitivity, literally from any plant tissue. Additionally, this method can be used to confirm the size, stability and the abundance of miRNAs and their precursors.

This method demonstrates use of the northern hybridization technique to detect miRNAs from total RNA extract.

Bitki MikroRNAlarının Saptanması ve Ölçülmesi için RNA Leke Analizi

MicroRNAs (miRNAs) are a class of endogenously expressed non-coding, ~21 nt small RNAs involved in the regulation of gene expression in both plants and ...