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<ol>
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A., Freire, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+protein-protein+interactions+by+isothermal+titration+calorimetry.">Characterization of protein-protein interactions by isothermal titration calorimetry.</a> Methods Mol. Biol. 261, 35-54 (2004).</li><li>Turnbull, W. B., Daranas, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+value+of+c:+can+low+affinity+systems+be+studied+by+isothermal+titration+calorimetry.">On the value of c: can low affinity systems be studied by isothermal titration calorimetry.</a> J. Am. Chem. Soc. 125, 14859-14866 (2003).</li><li>Fukada, H., Takahashi, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enthalpy+and+heat+capacity+changes+for+the+proton+dissociation+of+various+buffer+components+in+0.1+M+potassium+chloride.">Enthalpy and heat capacity changes for the proton dissociation of various buffer components in 0.1 M potassium chloride.</a> Proteins. 33, 159-166 (1998).</li><li>Houtman, J. 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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unusual+binding+stoichiometries+and+cooperativity+are+observed+during+binary+and+ternary+complex+formation+in+the+single+active+pore+of+R67+dihydrofolate+reductase,+a+D2+symmetric+protein.">Unusual binding stoichiometries and cooperativity are observed during binary and ternary complex formation in the single active pore of R67 dihydrofolate reductase, a D2 symmetric protein.</a> Biochemistry. 35, 11414-11424 (1996).</li><li>Shenoy, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multisite+and+multivalent+binding+between+cyanovirin-N+and+branched+oligomannosides:+calorimetric+and+NMR+characterization.">Multisite and multivalent binding between cyanovirin-N and branched oligomannosides: calorimetric and NMR characterization.</a> Chem. Biol. 9, 1109-1118 (2002).</li><li>Chopra, S., Lynch, R., Kim, S. H., Jackson, M., Howell, E. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+temperature+and+viscosity+on+R67+dihydrofolate+reductase+catalysis.">Effects of temperature and viscosity on R67 dihydrofolate reductase catalysis.</a> Biochemistry. 45, 6596-6605 (2006).</li><li>Norris, A. L., Serpersu, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactions+of+Coenzyme+A+with+the+Aminoglycoside+Acetyltransferase+(3)-IIIb+and+Thermodynamics+of+a+Ternary+System.">Interactions of Coenzyme A with the Aminoglycoside Acetyltransferase (3)-IIIb and Thermodynamics of a Ternary System.</a> Biochemistry. 49, 4036-4042 (2010).</li><li>Falconer, R. J., Penkova, A., Jelesarov, I., Collins, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Survey+of+the+year+2008:+applications+of+isothermal+titration+calorimetry.">Survey of the year 2008: applications of isothermal titration calorimetry.</a> J. Mol. Recognit. 23, 395-413 (2010).</li><li>Ababou, A., Ladbury, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Survey+of+the+year+2005:+literature+on+applications+of+isothermal+titration+calorimetry.">Survey of the year 2005: literature on applications of isothermal titration calorimetry.</a> J. Mol. Recognit. 20, 4-14 (2007).</li><li>Buurma, N. J., Haq, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+the+analysis+of+isothermal+titration+calorimetry+data+for+ligand-DNA+interactions.">Advances in the analysis of isothermal titration calorimetry data for ligand-DNA interactions.</a> Methods. 42, 162-172 (2007).</li><li>Salim, N. N., Feig, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isothermal+titration+calorimetry+of+RNA.">Isothermal titration calorimetry of RNA.</a> Methods. 47, 198-205 (2009).</li><li>De, M., You, C. C., Srivastava, S., Rotello, V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomimetic+interactions+of+proteins+with+functionalized+nanoparticles:+a+thermodynamic+study.">Biomimetic interactions of proteins with functionalized nanoparticles: a thermodynamic study.</a> J. Am. Chem. Soc. 129, 10747-10753 (2007).</li><li>Velazquez-Campoy, A., Freire, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isothermal+titration+calorimetry+to+determine+association+constants+for+high-affinity+ligands.">Isothermal titration calorimetry to determine association constants for high-affinity ligands.</a> Nat. Protoc. 1, 186-191 (2006).</li><li>Velazquez Campoy, A., Freire, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ITC+in+the+post-genomic+era...?+Priceless.">ITC in the post-genomic era...? Priceless.</a> Biophys. Chem. 115, 115-124 (2005).</li><li>Harries, D., Rau, D. C., Parsegian, V. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Solutes+probe+hydration+in+specific+association+of+cyclodextrin+and+adamantane.">Solutes probe hydration in specific association of cyclodextrin and adamantane.</a> J. Am. Chem. Soc. 127, 2184-2190 (2005).</li><li>Chervenak, M. C., Toone, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+direct+measure+of+the+contribution+of+solvent+reorganization+to+the+enthalpy+of+binding.">A direct measure of the contribution of solvent reorganization to the enthalpy of binding.</a> J. Am. Chem. Soc. 116, 10533-10539 (1994).</li><li>Taneva, S. G., Moro, F., Velazquez-Campoy, A., Muga, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Energetics+of+nucleotide-induced+DnaK+conformational+states.">Energetics of nucleotide-induced DnaK conformational states.</a> Biochemistry. 49, 1338-1345 (2010).</li><li>Jelesarov, I., Bosshard, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermodynamics+of+ferredoxin+binding+to+ferredoxin:NADP++reductase+and+the+role+of+water+at+the+complex+interface.">Thermodynamics of ferredoxin binding to ferredoxin:NADP+ reductase and the role of water at the complex interface.</a> Biochemistry. 33, 13321-13328 (1994).</li><li>Burrows, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+the+monomer-dimer+equilibrium+of+interleukin-8+reveals+it+is+a+monomer+at+physiological+concentration.">Determination of the monomer-dimer equilibrium of interleukin-8 reveals it is a monomer at physiological concentration.</a> Biochemistry. 33, 12741-12745 (1994).</li><li>Baker, B. M., Murphy, K. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+linked+protonation+effects+in+protein+binding+reactions+using+isothermal+titration+calorimetry.">Evaluation of linked protonation effects in protein binding reactions using isothermal titration calorimetry.</a> Biophys. J. 71, 2049-2055 (1996).</li></ol>

利害の衝突は宣言されません。

ITCは、11は蛋白質-リガンド、12 DNA -配位子13とRNA -高分子を見て研究14の研究で、リガンド高分子の相互作用の研究に広く使用されています。 ITCは、さらに均一な懸濁液を形成するようなナノ粒子等の固体材料を、、で実行することができます。つのリガンドが既に巨大分子と第2のリガンドに結合している15さらに、三元系システムは、滴定、熱力学を決定するために使用することができる、のために弱い結合の例、酵素補因子のシステムへの基質の結合7研究も通常より弱い結合リガンドとの競合結合アッセイを行うことにより、ITCの検出限界を超えてしまう非常に高い結合親和性を持つ分子に対して実行することができます。16情報リガンドはまた、競合アッセイによって得ることができます。17が結合における水の役割は、溶媒の再編成に関するエンタルピーの依存性とともにITC、18によって探索することができます。19は最近、DNAK変異体のコンフォメーション変化の熱力学があるのは結合時に測定したADPとATP 20タンパク質-タンパク質の関連付けはITCが調査することができる、ホモ関連で同様のヘテロ複合体に関する情報21を得た。結合エンタルピーで22温度の効果は結合事象の熱容量を与える。2の数結合時に吸収または放出陽子はまた、イオン化の異なるエンタルピーとバッファで行われた実験から決定することができます。5つの追加ITCの研究は、異なるpH値で行われている場合は、関係するグループのpKaは潜在的に決定することができます。23 要約すると、高分子とリガンドの濃度の正確な測定が良いITCの実験のための不可欠です。サンプルの濃度はまた、信頼性の高いデータを得るために適切な範囲内にする必要があります。小分子の不純物とpHの不一致は、サーモグラムでアーティファクトが発生するので注意がバッファを用いる場合には注意が必要。

<table border="1"><tbody><tr><th>試薬の名前</th><th>会社</th><th>カタログ番号</th></tr><tr><td>を1.7 mLマイクロ遠心チューブ</td><td>フィッシャーサイエンティフィック</td><td> 02-681-282 </td></tr><tr><td> 15 mLのファルコンチューブ</td><td>フィッシャーサイエンティフィック</td><td> 14から959 - 49B </td></tr><tr><td> 2.5mLのハミルトンシリンジ</td><td> MicroCal </td><td> SYN161714 </td></tr></tbody></table>

isothermal titration calorimetry for measuring macromolecule-ligand affinity

等温滴定熱量測定（ITC）は、結合反応の完全な熱力学の絵を理解するための便利なツールです。生物学では、高分子の相互作用は、細胞の機械を理解する上で不可欠です。このようなバッファや温度などExperimental条件は、研究対象の特定のバインディングのシステムに合わせて調整することができます。特定のリガンドと高分子濃度範囲は、有用なデータを取得するために必要なので、しかし、慎重な計画が必要です。高分子とリガンドの濃度は正確に信頼性の高い結果を得るために決定する必要があります。ケアにも不純物が大幅に実験に影響を与えることができるようにサンプルを準備する際に注意が必要。このような化学量論、親和性やエンタルピーなどのコントロールと一緒にITCの実験は、、適切に実行される、有用なバインディング情報は、得られる。別のバッファまたは温度条件下での追加実験を実行することによって、より詳細な情報は、システムについて取得することができます。 ITCの実験の基本的なセットアップのためのプロトコルが与えられます。

Watch this Scientific Journal Video about Isothermal Titration Calorimetry for Measuring Macromolecule-Ligand Affinity at JoVE.com

Isothermal Titration Calorimetry for Measuring Macromolecule-Ligand Affinity

中程度の結合親和性と生物系のための結合熱力学を監視するために等温滴定熱量計を使用するための一般的なプロトコルが表示されます。

高分子 - リガンド親和性を測定するための等温滴定カロリメトリー

Isothermal titration calorimetry (ITC) is a useful tool for understanding the complete thermodynamic picture of a binding reaction.  In biological ...

isothermal-titration-calorimetry-for-measuring-macromolecule-ligand

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Unit 1

Acid–Base Titration

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52.5K ビュー.  University of Tennessee. 中程度の結合親和性と生物系のための結合熱力学を監視するために等温滴定熱量計を使用するための一般的なプロトコルが表示されます。

ビデオ: Isothermal Titration Calorimetry for Measuring Macromolecule-Ligand Affinity

Titration of Human Coronaviruses Using an Immunoperoxidase Assay

Determination of infectious viral titers is a basic and essential experimental approach for virologists. Classical plaque assays cannot be used for viruses that do not cause significant cytopathic effects, which is the case for prototype strains 229E and OC43 of human coronavirus (HCoV). Therefore, an alternative indirect immunoperoxidase assay (IPA) was developed for the detection and titration of these viruses and is described herein. Susceptible cells are inoculated with serial logarithmic dilutions of virus-containing samples in a 96-well plate format. After viral growth, viral detection by IPA yields the infectious virus titer, expressed as 'Tissue Culture Infectious Dose 50 percent' (TCID50). This represents the dilution of a virus-containing sample at which half of a series of laboratory wells contain infectious replicating virus. This technique provides a reliable method for the titration of HCoV-229E and HCoV-OC43 in biological samples such as cells, tissues and fluids. This article is based on work first reported in Methods in Molecular Biology (2008) volume 454, pages 93-102.

In this video, we demonstrate an alternative method for detection and titering of viruses using an enzymatic antigen detection technique known as an immunoperoxidase assay. Here, we will show you how to collect your viral samples, prepare the cells for testing, and finally the immunoperoxidase assay using serial dilutions to determine the viral titer.

免疫ペルオキシダーゼアッセイを用いたヒトコロナウイルスの滴定

Determination of infectious viral titers is a basic and essential experimental approach for virologists. Classical plaque assays cannot be used for ...

生物学

Collecting Variable-concentration Isothermal Titration Calorimetry Datasets in Order to Determine Binding Mechanisms

Isothermal titration calorimetry (ITC) is commonly used to determine the thermodynamic parameters associated with the binding of a ligand to a host macromolecule. ITC has some advantages over common spectroscopic approaches for studying host/ligand interactions. For example, the heat released or absorbed when the two components interact is directly measured and does not require any exogenous reporters. Thus the binding enthalpy and the association constant (Ka) are directly obtained from ITC data, and can be used to compute the entropic contribution. Moreover, the shape of the isotherm is dependent on the c-value and the mechanistic model involved. The c-value is defined as c = n[P]tKa, where [P]t is the protein concentration, and n is the number of ligand binding sites within the host. In many cases, multiple binding sites for a given ligand are non-equivalent and ITC allows the characterization of the thermodynamic binding parameters for each individual binding site. This however requires that the correct binding model be used. This choice can be problematic if different models can fit the same experimental data. We have previously shown that this problem can be circumvented by performing experiments at several c-values. The multiple isotherms obtained at different c-values are fit simultaneously to separate models. The correct model is next identified based on the goodness of fit across the entire variable-c dataset. This process is applied here to the aminoglycoside resistance-causing enzyme aminoglycoside N-6'-acetyltransferase-Ii (AAC(6')-Ii). Although our methodology is applicable to any system, the necessity of this strategy is better demonstrated with a macromolecule-ligand system showing allostery or cooperativity, and when different binding models provide essentially identical fits to the same data. To our knowledge, there are no such systems commercially available. AAC(6')-Ii, is a homo-dimer containing two active sites, showing cooperativity between the two subunits. However ITC data obtained at a single c-value can be fit equally well to at least two different models a two-sets-of-sites independent model and a two-site sequential (cooperative) model. Through varying the c-value as explained above, it was established that the correct binding model for AAC(6')-Ii is a two-site sequential binding model. Herein, we describe the steps that must be taken when performing ITC experiments in order to obtain datasets suitable for variable-c analyses.

ITC is a powerful tool for studying the binding of a ligand to its host. In complex systems however, several models may fit the data equally well. The method described here provides a means to elucidate the appropriate binding model for complex systems and extract the corresponding thermodynamic parameters.

バインディング機構を決定するために、可変濃度等温滴定熱量計のデータセットの収集

Isothermal titration calorimetry (ITC) is commonly used to determine the thermodynamic parameters associated with the binding of a ligand to a host ...

High-throughput Purification of Affinity-tagged Recombinant Proteins

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further biochemical analyses to obtain detailed insights into structure/function relationships. Advances in oligonucleotide- and gene synthesis technology make large-scale mutagenesis strategies increasingly feasible, including the substitution of target residues by all 19 other amino acids. Gain- or loss-of-function phenotypes then allow systematic conclusions to be drawn, such as the contribution of particular residues to catalytic activity, protein stability and/or protein-protein interaction specificity.
In order to attribute the different phenotypes to the nature of the mutation - rather than to fluctuating experimental conditions - it is vital to purify and analyse the proteins in a controlled and reproducible manner. High-throughput strategies and the automation of manual protocols on robotic liquid-handling platforms have created opportunities to perform such complex molecular biological procedures with little human intervention and minimal error rates1-5.
Here, we present a general method for the purification of His-tagged recombinant proteins in a high-throughput manner. In a recent study, we applied this method to a detailed structure-function investigation of TFIIB, a component of the basal transcription machinery. TFIIB is indispensable for promoter-directed transcription in vitro and is essential for the recruitment of RNA polymerase into a preinitiation complex6-8. TFIIB contains a flexible linker domain that penetrates the active site cleft of RNA polymerase9-11. This linker domain confers two biochemically quantifiable activities on TFIIB, namely (i) the stimulation of the catalytic activity during the 'abortive' stage of transcript initiation, and (ii) an additional contribution to the specific recruitment of RNA polymerase into the preinitiation complex4,5,12 . We exploited the high-throughput purification method to generate single, double and triple substitution and deletions mutations within the TFIIB linker and to subsequently analyse them in functional assays for their stimulation effect on the catalytic activity of RNA polymerase4. Altogether, we generated, purified and analysed 381 mutants - a task which would have been time-consuming and laborious to perform manually. We produced and assayed the proteins in multiplicates which allowed us to appreciate any experimental variations and gave us a clear idea of the reproducibility of our results.
This method serves as a generic protocol for the purification of His-tagged proteins and has been successfully used to purify other recombinant proteins. It is currently optimised for the purification of 24 proteins but can be adapted to purify up to 96 proteins.

We describe a method for the affinity-tagged purification of recombinant proteins using liquid-handling robotics. This method is generally applicable to the small-scale purification of soluble His-tagged proteins in a high-throughput format.

アフィニティータグ組換えタンパク質のハイスループット精製

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further ...

Biochemical Titration of Glycogen In vitro

Glycogen is the main energetic polymer of glucose in vertebrate animals and plays a crucial role in whole body metabolism as well as in cellular metabolism. Many methods to detect glycogen already exist but only a few are quantitative. We describe here a method using the Abcam Glycogen assay kit, which is based on specific degradation of glycogen to glucose by glucoamylase. Glucose is then specifically oxidized to a product that reacts with the OxiRed probe to produce fluorescence. Titration is accurate, sensitive and can be achieved on cell extracts or tissue sections. However, in contrast to other techniques, it does not give information about the distribution of glycogen in the cell. As an example of this technique, we describe here the titration of glycogen in two cell lines, Chinese hamster lung fibroblast CCL39 and human colon carcinoma LS174, incubated in normoxia (21% O2) versus hypoxia (1% O2). We hypothesized that hypoxia is a signal that prepares cells to synthesize and store glycogen in order to survive1.

Biochemical Titration of Glycogen In vitro

We describe here an accurate, reproducible and convenient biochemical method for titration of glycogen in vitro. This technique uses the Abcam Glycogen assay kit and is based on successive hydrolysis of glycogen to glucose and glucose titration by fluorescence.

グリコーゲンの生化学的滴定<em&gt;インビトロ</em

Glycogen is the main energetic polymer of glucose in vertebrate animals and plays a crucial role in whole body metabolism as well as in cellular ...

A Protocol for Phage Display and Affinity Selection Using Recombinant Protein Baits

Using recombinant phage as a scaffold to present various protein portions encoded by a directionally cloned cDNA library to immobilized bait molecules is an efficient means to discover interactions. The technique has largely been used to discover protein-protein interactions but the bait molecule to be challenged need not be restricted to proteins. The protocol presented here has been optimized to allow a modest number of baits to be screened in replicates to maximize the identification of independent clones presenting the same protein. This permits greater confidence that interacting proteins identified are legitimate interactors of the bait molecule. Monitoring the phage titer after each affinity selection round provides information on how the affinity selection is progressing as well as on the efficacy of negative controls. One means of titering the phage, and how and what to prepare in advance to allow this process to progress as efficiently as possible, is presented. Attributes of amplicons retrieved following isolation of independent plaque are highlighted that can be used to ascertain how well the affinity selection has progressed. Trouble shooting techniques to minimize false positives or to bypass persistently recovered phage are explained. Means of reducing viral contamination flare up are discussed.

Phage display is a powerful technique to capture proteins or protein moieties that interact with an immobilized molecule of interest. Once a decision of the type of phage cDNA library to create and screen has been made, the protocol described here permits efficient affinity selection leading to identification of interactors.

組換えタンパク質ベイトを用いてファージディスプレイおよび親和性選択のためのプロトコル

Using  recombinant phage as a scaffold to present various protein portions encoded by  a directionally cloned cDNA library to immobilized bait molecules ...

Affinity-based Isolation of Tagged Nuclei from Drosophila Tissues for Gene Expression Analysis

Drosophila melanogaster embryonic and larval tissues often contain a highly heterogeneous mixture of cell types, which can complicate the analysis of gene expression in these tissues. Thus, to analyze cell-specific gene expression profiles from Drosophila tissues, it may be necessary to isolate specific cell types with high purity and at sufficient yields for downstream applications such as transcriptional profiling and chromatin immunoprecipitation. However, the irregular cellular morphology in tissues such as the central nervous system, coupled with the rare population of specific cell types in these tissues, can pose challenges for traditional methods of cell isolation such as laser microdissection and fluorescence-activated cell sorting (FACS). Here, an alternative approach to characterizing cell-specific gene expression profiles using affinity-based isolation of tagged nuclei, rather than whole cells, is described. Nuclei in the specific cell type of interest are genetically labeled with a nuclear envelope-localized EGFP tag using the Gal4/UAS binary expression system. These EGFP-tagged nuclei can be isolated using antibodies against GFP that are coupled to magnetic beads. The approach described in this protocol enables consistent isolation of nuclei from specific cell types in the Drosophila larval central nervous system at high purity and at sufficient levels for expression analysis, even when these cell types comprise less than 2% of the total cell population in the tissue. This approach can be used to isolate nuclei from a wide variety of Drosophila embryonic and larval cell types using specific Gal4 drivers, and may be useful for isolating nuclei from cell types that are not suitable for FACS or laser microdissection.

Affinity-based Isolation of Tagged Nuclei from Drosophila Tissues for Gene Expression Analysis

Drosophila tissues often contain a heterogeneous mixture of cell types.&#160;To examine gene expression in specific cell types from a particular tissue, nuclei can be genetically tagged and subsequently isolated using an affinity-based approach. Isolated nuclei can be used for downstream applications such as gene expression analysis and chromatin immunoprecipitation.

からのタグ付き核の親和性に基づく分離<em&gt;ショウジョウバエ</em遺伝子発現解析のための&gt;組織

Drosophila melanogaster embryonic and larval tissues often contain a highly heterogeneous mixture of cell types, which can complicate the analysis of gene ...

High-throughput Titration of Luciferase-expressing Recombinant Viruses

Standard plaque assays to determine infectious viral titers can be time consuming, are not amenable to a high volume of samples, and cannot be done with viruses that do not form plaques. As an alternative to plaque assays, we have developed a high-throughput titration method that allows for the simultaneous titration of a high volume of samples in a single day. This approach involves infection of the samples with a Firefly luciferase tagged virus, transfer of the infected samples onto an appropriate permissive cell line, subsequent addition of luciferin, reading of plates in order to obtain luminescence readings, and finally the conversion from luminescence to viral titers. The assessment of cytotoxicity using a metabolic viability dye can be easily incorporated in the workflow in parallel and provide valuable information in the context of a drug screen. This technique provides a reliable, high-throughput method to determine viral titers as an alternative to a standard plaque assay.

This article presents a high-throughput luciferase expression-based method of titrating various RNA and DNA viruses using automated and manual liquid handlers.

ルシフェラーゼを発現する組換えウイルスの高スループット滴定

Standard plaque assays to determine infectious viral titers can be time consuming, are not amenable to a high volume of samples, and cannot be done with ...

Purification of Native Complexes for Structural Study Using a Tandem Affinity Tag Method

Affinity purification approaches have been successful in isolating native complexes for proteomic characterization. Structural heterogeneity and a degree of compositional heterogeneity of a complex do not usually impede progress in conducting such studies. In contrast, a complex intended for structural characterization should be purified in a state that is both compositionally and structurally homogeneous as well as at a higher concentration than required for proteomics. Recently, there have been significant advances in the application of electron microscopy for structure determination of large macromolecular complexes. This has heightened interest in approaches to purify native complexes of sufficient quality and quantity for structural determination by electron microscopy. The Tandem Affinity Purification (TAP) method has been optimized to extract and purify an 18-subunit, ~ 0.8 MDa ribonucleoprotein assembly from budding yeast (Saccharomyces cerevisiae) suitable for negative stain and electron cryo microscopy. Herein is detailed the modifications made to the TAP method, the rationale for making these changes, and the approaches taken to assay for a compositionally and structurally homogeneous complex.

The Tandem Affinity Purification (TAP) method has been used extensively to isolate native complexes from cellular extract, primarily eukaryotic, for proteomics. Here, we present a TAP method protocol optimized for purification of native complexes for structural studies.

タンデムアフィニティータグ法を用いた構造解析のためのネイティブ複合体の精製

Affinity purification approaches have been successful in isolating native complexes for proteomic characterization. Structural heterogeneity and a degree ...

A Customizable Chamber for Measuring Cell Migration

Cell migration is a vital part of immune responses, growth, and wound healing. Cell migration is a complex process that involves interactions between cells, the extracellular matrix, and soluble and non-soluble chemical factors (e.g., chemoattractants). Standard methods for measuring the migration of cells, such as the Boyden chamber assay, work by counting cells on either side of a divider. These techniques are easy to use; however, they offer little geometric modification for different applications. In contrast, microfluidic devices can be used to observe cell migration with customizable concentration gradients of soluble factors1,2. However, methods for making microfluidics based assays can be difficult to learn.
Here, we describe an easy method for creating cell culture chambers to measure cell migration in response to chemical concentration gradients. Our cell migration chamber method can create different linear concentration gradients in order to study cell migration for a variety of applications. This method is relatively easy to use and is typically performed by undergraduate students.
The microchannel chamber was created by placing an acrylic insert in the shape of the final microchannel chamber well into a Petri dish. After this, poly(dimethylsiloxane) (PDMS) was poured on top of the insert. The PDMS was allowed to harden and then the insert was removed. This allowed for the creation of wells in any desired shape or size. Cells may be subsequently added to the microchannel chamber, and soluble agents can be added to one of the wells by soaking an agarose block in the desired agent. The agarose block is added to one of the wells, and time-lapse images can be taken of the microchannel chamber in order to quantify cell migration. Variations to this method can be made for a given application, making this method highly customizable.

This protocol details a customizable method to measure cell migration in response to chemoattractants that may also be used to determine the diffusion rate of a drug out of a polymer matrix.

細胞移動を測定するためのカスタマイズ可能な商工会議所

Cell migration is a vital part of immune responses, growth, and wound healing. Cell migration is a complex process that involves interactions between ...

Simultaneous Affinity Enrichment of Two Post-Translational Modifications for Quantification and Site Localization

Studying multiple post-translational modifications (PTMs) of proteins is a crucial step to understand PTM crosstalk and gain more holistic insights into protein function. Despite the importance of multi-PTM enrichment studies, few studies investigate more than one PTM at a time, due partially to the expenses, time, and large protein quantities required to perform multiple global proteomic analysis of PTMs. The &#34;one-pot&#34; affinity enrichment detailed in this protocol overcomes these barriers by permitting the simultaneous identification and quantification of peptides with lysine residues containing acetylation and succinylation PTMs with low amounts of sample input. The protocol involves preparation of protein lysate from mouse livers of SIRT5 knockout mice, performance of trypsin digestion, enrichment for PTMs, and performance of mass spectrometric analysis using a data-independent acquisition (DIA) workflow. Because this workflow allows for the enrichment of two PTMs from the same sample simultaneously, it provides a practical tool to study PTM crosstalk without requiring large amounts of samples, and it greatly reduces the time required for sample preparation, data acquisition, and analysis. The DIA component of the workflow provides comprehensive PTM-specific information. This is particularly important when studying PTM site localization, as DIA provides comprehensive sets of fragment ions that can be computationally deciphered to differentiate between different PTM localization isoforms.

This workflow describes the performance of time- and cost-efficient enrichment of multiple protein post-translational modifications (PTMs) simultaneously for quantitative global proteomic analysis. The protocol utilizes peptide-level PTM enrichment with multiple conjugated antibodies, followed by data-independent acquisition mass spectrometry analysis to gain biological insights into PTM crosstalk.

定量化とサイトローカリゼーションのための2つの翻訳後修飾の同時アフィニティエンリッチメント

Studying multiple post-translational modifications (PTMs) of proteins is a crucial step to understand PTM crosstalk and gain more holistic insights into ...