この作品は、国立ニードプログラム（P200A100044）の分野で大学院支援を通じて教育省によってサポートされていました。

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著者らは、開示することは何もありません。

我々が説明したニトロセルロース結合アッセイは、ヒスチジンキナーゼを特徴付ける以前に使用されている方法に比べて多くの利点を有しています。従来のSDS / PAGEに基づくオートラジオグラフィーと比較して、我々の方法は、より高いスループットおよびより少ない時間がかかります。ニトロセルロース膜は、SDSゲルよりも取り扱いが容易であり、かつ固定される必要はありません。タンパ...

適応応答は、細菌の生存のために重要です。検出し、環境変化に対応するためには、細菌は、二成分情報伝達として知られている刺激応答システムを使用しています。典型的な2成分系では1,2は 、ヒスチジンキナーゼは、同族の刺激を検出し、その保存されたヒスチジン残基を自己リン酸化し、その後、応答調節タンパク質のレシーバ・ドメインに保存されたアスパラギン残基にリン酸を転送します。 3このイベントは、下流の効果を刺激するレスポンスレギュレーターの活性の変化を誘発します。 4,5したがって 、細菌は、センスおよびローカル環境の変化に適応することができます。いくつかの二成分シグナリングシステムは、この原型から逸脱します。いくつかのケースでは、ヒスチジンキナーゼの感覚ドメインに直接感覚入力を検出し、タンパク質 - タンパク質相互作用を介してキナーゼ活性を修飾するスタンドアロンのタンパク質です。しかし8、ファンド- 6amentalプロセスおよびシステムの全体的な役割は変わりません。二成分シグナル伝達は、細菌の生存に必須であるユビキタス刺激応答システムであって、ヒスチジンキナーゼは、シグナルの伝達に重要な役割を果たしています。 9 細菌の生物学へのヒスチジンキナーゼの重要性にもかかわらず、彼らは特徴付けが乏しいまま。これは、ホスホの固有の不安定性、および自己リン酸化を測定するための実用的な方法が不足しているためです。ホスホはホスホセリン、ホスホスレオニン、およびホスホチロシンよりも不安定です。 10はこのように、一般にセリン/スレオニン/ Tyrのキナーゼを分析するために使用されている技術は、ヒスチジンキナーゼには適用されません。ヒスチジンキナーゼを研究するためのインビトロアッセイにおいて 、11は、主に SDS-PAGEオートラジオグラフィーに限られていました。 12,13この方法では、[γ-32 P] -ATPをキナーゼとインキュベートし、キナーゼのリン酸化がPOLによって分析されますyacrylamideゲル電気泳動（PAGE）は、ゲルのオートラジオグラフィーを行いました。この方法は、キナーゼの自己リン酸化、ならびにキナーゼからの応答レギュレーターのリン酸転移をモニターするために使用することができます。しかし、この方法では、顕著な欠点があります。ページベースのアッセイは、ロースループットおよび時間がかかります。このような制限は、タンパク質を特徴付けるし、その動態パラメータを確認に資するではありません。最近公開されたヒスチジンキナーゼを研究するための別の方法は、自己リン酸化を検出するためにホスホ抗体を利用します。 14この方法は、検出のために使用した測定方法に応じて、1-ホスホおよび3-ホスホとを区別するという利点を有しているが、この方法は、大きなダイナミックレンジまたは検出の高い上限を提供しない場合があります。したがって、これらの重要なタンパク質を研究するために使用することができるより速く、より少ない労力であり、より高感度のアッセイが必要とされています。 ここでは、説明すると、dインビトロでの精製の細菌ヒスチジンキナーゼの自己リン酸化を定量化するために使用することができる慎重に開発され、ニトロセルロース結合アッセイをemonstrate。このアッセイは、より高いスループットおよびPAGEベースのアッセイよりも時間がかかります。この方法はまた、検出と大きなダイナミックレンジの高い上限を提供していますホスホ定量化のためのチェレンコフ放射を利用します。アッセイは、ヒスチジンキナーゼの動力学的パラメーターを決定するために用いることができます。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Phosphoric acid</td>
			<td>VWR</td>
			<td>AAAA18067-AP</td>
			<td>For quenching reactions and washing nitrocellulose</td>
		</tr>
		<tr>
			<td>Tris base</td>
			<td>RPI</td>
			<td>T60040-5000.0</td>
			<td>Tris-HCl pH 8.0, for kinase reaction buffer</td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>RPI</td>
			<td>P41000-2500.0</td>
			<td>For kinase reaction buffer</td>
		</tr>
		<tr>
			<td>Magnesium chloride</td>
			<td>RPI</td>
			<td>M24000-500.0</td>
			<td>For kinase reaction buffer</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>RPI</td>
			<td>G22020-4000.0</td>
			<td>For kinase reaction buffer</td>
		</tr>
		<tr>
			<td>5&#39;-ATP</td>
			<td>Promega</td>
			<td>E6011</td>
			<td>Kinase substrate</td>
		</tr>
		<tr>
			<td>[&#947;-32P]-5&#39;-ATP</td>
			<td>Perkin Elmer</td>
			<td>NEG002Z250UC&#160;</td>
			<td>6000 Ci/mmol</td>
		</tr>
		<tr>
			<td>96-well dot blot apparatus</td>
			<td>Bio-rad</td>
			<td>1706545</td>
			<td>For spotting reactions</td>
		</tr>
		<tr>
			<td>Nitrocellulose</td>
			<td>Whatman</td>
			<td>32-10401396-PK</td>
			<td>For spotting reactions</td>
		</tr>
		<tr>
			<td>Ponceau S</td>
			<td>Sigma aldrich</td>
			<td>P3504-50G</td>
			<td>For staining nitrocellulose</td>
		</tr>
	</tbody>
</table>

quantification of bacterial histidine kinase autophosphorylation using a nitrocellulose binding assay

We demonstrate a useful method for quantifying autophosphorylation of purified bacterial histidine kinases. Histidine kinases are known for their involvement in two-component signal transduction, a ubiquitous system through which bacteria sense and respond to environmental stimuli. Two-component signaling features autophosphorylation of a histidine kinase, followed by phosphotransfer to the receiver domain of a response regulator protein, which ultimately leads to an output response. Autophosphorylation of the histidine kinase is responsive to the presence of a cognate environmental stimulus, thereby giving bacteria a means to detect and respond to changes in the environment. Despite their importance in bacterial biology, histidine kinases remain poorly understood due to the inherent lability of phosphohistidine. Conventional methods for studying these proteins, such as SDS-PAGE autoradiography, have significant shortcomings. We have developed a nitrocellulose binding assay that can be used to characterize histidine kinases. The protocol for this assay is simple and easy to execute. Our method is higher throughput, less time-consuming, and offers a greater dynamic range than SDS-PAGE autoradiography.

<html>代表的なデータセットは、蛍光イメージャー（ 図1）ニトロセルロース膜を、ポンソーS染色（ 図2）、およびシンチレーション計数データ（ 図3）で撮影した画像の特徴を生成しました。 図3Aは、ラインウィーバー・バークプロット中の酵素反応速度定数を示しています。これらの結果は、グラム陰性種腸炎ビ...

Watch this Scientific Journal Video about Quantification of Bacterial Histidine Kinase Autophosphorylation Using a Nitrocellulose Binding Assay at JoVE.com

Quantification of Bacterial Histidine Kinase Autophosphorylation Using a Nitrocellulose Binding Assay

We report and demonstrate an optimized nitrocellulose binding assay that can be used to quantify autophosphorylation of purified bacterial histidine kinases. Our method has several advantages over traditional SDS-PAGE based techniques, providing a valuable alternative for characterizing these important proteins.

ニトロセルロース結合アッセイを用いて細菌ヒスチジンキナーゼ自己リン酸化の定量化

We demonstrate a useful method for quantifying autophosphorylation of purified bacterial histidine kinases. Histidine kinases are known for their ...

quantification-bacterial-histidine-kinase-autophosphorylation-using

Universidad de Cartagena UDEC

Research

JoVE Journal

Biochemistry

8.5K ビュー.  Stony Brook University. We report and demonstrate an optimized nitrocellulose binding assay that can be used to quantify autophosphorylation of purified bacterial histidine kinases. Our method has several advantages over traditional SDS-PAGE based techniques, providing a valuable alternative for characterizing these important proteins.

ビデオ: Quantification of Bacterial Histidine Kinase Autophosphorylation Using a Nitrocellulose Binding Assay

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.

共沈降によるF-アクチンへのタンパク質結合の測定

Filamentous actin (F-actin) organization within cells is regulated by a large number of actin-binding proteins that control actin nucleation, growth, ...

生化学

Rab10 Phosphorylation Detection by LRRK2 Activity Using SDS-PAGE with a Phosphate-binding Tag

Mutations in leucine-rich repeat kinase 2 (LRRK2) have been shown to be linked with familial Parkinson&#39;s disease (FPD). Since abnormal activation of the kinase activity of LRRK2 has been implicated in the pathogenesis of PD, it is essential to establish a method to evaluate the physiological levels of the kinase activity of LRRK2. Recent studies revealed that LRRK2 phosphorylates members of the Rab GTPase family, including Rab10, under physiological conditions. Although the phosphorylation of endogenous Rab10 by LRRK2 in cultured cells could be detected by mass spectrometry, it has been difficult to detect it by immunoblotting due to the poor sensitivity of currently available phosphorylation-specific antibodies for Rab10. Here, we describe a simple method of detecting the endogenous levels of Rab10 phosphorylation by LRRK2 based on immunoblotting utilizing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) combined with a phosphate-binding tag (P-tag), which is N-(5-(2-aminoethylcarbamoyl)pyridin-2-ylmetyl)-N,N&#39;,N&#39;-tris(pyridin-2-yl-methyl)-1,3-diaminopropan-2-ol. The present protocol not only provides an example of the methodology utilizing the P-tag but also enables the assessment of how mutations as well as inhibitor treatment/administration or any other factors alter the downstream signaling of LRRK2 in cells and tissues.

The present study describes a simple method of detecting endogenous levels of Rab10 phosphorylation by leucine-rich repeat kinase 2.

SDS ページを用いたリン酸 binding タグ LRRK2 活動によって Rab10 リン酸化検出

Mutations in leucine-rich repeat kinase 2 (LRRK2) have been shown to be linked with familial Parkinson&#39;s disease (FPD). Since abnormal activation of ...

Method for Efficient Refolding and Purification of Chemoreceptor Ligand Binding Domain

Identification of natural ligands of chemoreceptors and structural studies aimed at elucidation of the molecular basis of the ligand specificity can be greatly facilitated by the production of milligram amounts of pure, folded ligand binding domains. Attempts to heterologously express periplasmic ligand binding domains of bacterial chemoreceptors in Escherichia coli (E. coli) often result in their targeting into inclusion bodies. Here, a method is presented for protein recovery from inclusion bodies, its refolding and purification, using the periplasmic dCACHE ligand binding domain of Campylobacter jejuni (C. jejuni) chemoreceptor Tlp3 as an example. The approach involves expression of the protein of interest with a cleavable His6-tag, isolation and urea-mediated solubilisation of inclusion bodies, protein refolding by urea depletion, and purification by means of affinity chromatography, followed by tag removal and size-exclusion chromatography. The circular dichroism spectroscopy is used to confirm the folded state of the pure protein. It has been demonstrated that this protocol is generally useful for production of milligram amounts of dCACHE periplasmic ligand binding domains of other bacterial chemoreceptors in a soluble and crystallisable form.

A procedure is presented for the refolding of the dCACHE periplasmic ligand binding domain of Campylobacter jejuni chemoreceptor Tlp3 from inclusion bodies and the purification to yield milligram quantities of protein.

高効率巻き戻しと受容体リガンド結合領域の浄化法

Identification of natural ligands of chemoreceptors and structural studies aimed at elucidation of the molecular basis of the ligand specificity can be ...

Detection of Small GTPase Prenylation and GTP Binding Using Membrane Fractionation and  GTPase-linked Immunosorbent Assay

The Rho GTPase family belongs to the Ras superfamily and includes approximately 20 members in humans. Rho GTPases are important in the regulation of diverse cellular functions, including cytoskeletal dynamics, cell motility, cell polarity, axonal guidance, vesicular trafficking, and cell cycle control. Changes in Rho GTPase signaling play an essential regulatory role in many pathological conditions, such as&#160;cancer, central nervous system diseases, and immune system-dependent diseases. The posttranslational modification of Rho GTPases (i.e., prenylation by mevalonate pathway intermediates) and GTP binding are key factors which affect the activation of this protein. In this paper, two essential and simple methods are provided to detect a broad range of Rho GTPase prenylation and GTP binding activities. Details of the technical procedures that have been used are explained step by step in this manuscript.

Here we describe a protocol to investigate the prenylation and guanosine-5'-triphosphate (GTP)-loading of Rho GTPase. This protocol consists of two detailed methods, namely membrane fractionation and a GTPase-linked immunosorbent assay. The protocol can be used for measuring the prenylation and GTP loading of different other small GTPases.

小さい gtp アーゼ プレニル化および分子量 GTP 結合を使用して膜分画と gtp アーゼ抗体法の検出

The Rho GTPase family belongs to the Ras superfamily and includes approximately 20 members in humans. Rho GTPases are important in the regulation of ...

Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay

Cyclin-dependent kinase 1 (Cdk1) is a master controller for the cell cycle in all eukaryotes and phosphorylates an estimated 8 - 13% of the proteome; however, the number of identified targets for Cdk1, particularly in human cells is still low. The identification of Cdk1-specific phosphorylation sites is important, as they provide mechanistic insights into how Cdk1 controls the cell cycle. Cell cycle regulation is critical for faithful chromosome segregation, and defects in this complicated process lead to chromosomal aberrations and cancer.
Here, we describe an in vitro kinase assay that is used to identify Cdk1-specific phosphorylation sites. In this assay, a purified protein is phosphorylated in vitro by commercially available human Cdk1/cyclin B. Successful phosphorylation is confirmed by SDS-PAGE, and phosphorylation sites are subsequently identified by mass spectrometry. We also describe purification protocols that yield highly pure and homogeneous protein preparations suitable for the kinase assay, and a binding assay for the functional verification of the identified phosphorylation sites, which probes the interaction between a classical nuclear localization signal (cNLS) and its nuclear transport receptor karyopherin &#945;. To aid with experimental design, we review approaches for the prediction of Cdk1-specific phosphorylation sites from protein sequences. Together these protocols present a very powerful approach that yields Cdk1-specific phosphorylation sites and enables mechanistic studies into how Cdk1 controls the cell cycle. Since this method relies on purified proteins, it can be applied to any model organism and yields reliable results, especially when combined with cell functional studies.

Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay

Cyclin-dependent kinase 1 (Cdk1) is activated in the G2 phase of the cell cycle and regulates many cellular pathways. Here, we present a protocol for an in vitro kinase assay with Cdk1, which allows the identification of Cdk1-specific phosphorylation sites for establishing cellular targets of this important kinase.

サイクリン依存性キナーゼ 1 の同定の in Vitroキナーゼによって特定のリン酸化のサイトの試金します。

Cyclin-dependent kinase 1 (Cdk1) is a master controller for the cell cycle in all eukaryotes and phosphorylates an estimated 8 - 13% of the proteome; ...

Dissipative Microgravimetry to Study the Binding Dynamics of the Phospholipid Binding Protein Annexin A2 to Solid-supported Lipid Bilayers Using a Quartz Resonator

The dissipative quartz crystal microbalance technique is a simple and label-free approach to measure simultaneously the mass uptake and viscoelastic properties of the absorbed/immobilized mass on sensor surfaces, allowing the measurements of the interaction of proteins with solid-supported surfaces, such as lipid bilayers, in real-time and with a high sensitivity. Annexins are a highly conserved group of phospholipid-binding proteins that interact reversibly with the negatively charged headgroups via the coordination of calcium ions. Here, we describe a protocol that was employed to quantitatively analyze the binding of annexin A2 (AnxA2) to planar lipid bilayers prepared on the surface of a quartz sensor. This protocol is optimized to obtain robust and reproducible data and includes a detailed step-by-step description. The method can be applied to other membrane-binding proteins and bilayer compositions.

Here, we present an experimental protocol that can be employed to determine the binding affinities and mode of interaction of label-free phospholipid-binding protein annexin A2 with immobilized solid-supported bilayers (SLB) by simultaneously measuring the mass uptake and the viscoelastic properties of the protein annexin A2.

散逸の Microgravimetry 結合タンパク質アネキシン A2 への水晶振動子を用いた固相担脂質膜リン脂質の結合動態を研究するには

The dissipative quartz crystal microbalance technique is a simple and label-free approach to measure simultaneously the mass uptake and viscoelastic ...

Identification of Novel CK2 Kinase Substrates Using a Versatile Biochemical Approach

The study of kinase-substrate relationships is essential to gain a complete understanding of the functions of these enzymes and their downstream targets in both physiological and pathological states. CK2 is an evolutionarily conserved serine/threonine kinase with a growing list of hundreds of substrates involved in multiple cellular processes. Due to its pleiotropic properties, identifying and characterizing a comprehensive set of CK2 substrates has been particularly challenging and remains a hurdle in the study of this important enzyme. To address this challenge, we have devised a versatile experimental strategy that enables the targeted enrichment and identification of putative CK2 substrates. This protocol takes advantage of the unique dual co-substrate specificity of CK2 allowing for specific thiophosphorylation of its substrates in a cell or tissue lysate. These substrate proteins are subsequently alkylated, immunoprecipitated, and identified by liquid chromatography/tandem mass spectrometry (LC-MS/MS). We have previously used this approach to successfully identify CK2 substrates from Drosophila ovaries and here we extend the application of this protocol to human glioblastoma cells, illustrating the adaptability of this method to investigate the biological roles of this kinase in various model organisms and experimental systems.

The objective of this protocol is to label, enrich, and identify substrates of protein kinase CK2 from a complex biological sample such as a cell lysate or tissue homogenate. This method leverages unique aspects of CK2 biology for this purpose.

多彩な生化学的アプローチを用いた新規 CK2 キナーゼ基板の識別

The study of kinase-substrate relationships is essential to gain a complete understanding of the functions of these enzymes and their downstream targets ...

Characterization at the Molecular Level using Robust Biochemical Approaches of a New Kinase Protein

Extensive whole genome sequencing has identified many Open Reading Frames (ORFs) providing many potential proteins. These proteins may have important roles for the cell and may unravel new cellular processes. Among proteins, kinases are major actors as they belong to cell signaling pathways and have the ability to switch on or off many processes crucial to the fate of the cell, such as cell growth, division, differentiation, motility, and death.
In this study, we focused on a new potential kinase protein, LIMK2-1. We demonstrated its existence by Western Blot using a specific antibody. We evaluated its interaction with an upstream regulating protein using coimmunoprecipitation experiments. Coimmunoprecipitation is a very powerful technique able to detect the interaction between two target proteins. It may also be used to detect new partners of a bait protein. The bait protein may be purified either via a tag engineered to its sequence or via an antibody specifically targeting it. These protein complexes may then be separated by SDS-PAGE (Sodium Dodecyl Sulfate PolyAcrylamide Gel) and identified using mass spectrometry. Immunoprecipitated LIMK2-1 was also used to test its kinase activity in vitro by &#947;[32P] ATP labeling. This well-established assay may use many different substrates, and mutated versions of the bait may be used to assess the role of specific residues. The effects of pharmacological agents may also be evaluated since this technique is both highly sensitive and quantitative. Nonetheless, radioactivity handling requires particular caution. Kinase activity may also be assessed with specific antibodies targeting the phospho group of the modified amino acid. These kinds of antibodies are not commercially available for all the phospho modified residues.

We characterized a new kinase protein using robust biochemical approaches: Western Blot analysis with a dedicated specific antibody on different cell lines and tissues, interactions by coimmunoprecipitation experiments, kinase activity detected by Western Blot using a phospho-specific antibody and by &#947;[32P] ATP labeling.

新しいキナーゼタンパク質の堅牢な生化学的アプローチを用いて分子レベルでの特性解析

Extensive whole genome sequencing has identified many Open Reading Frames (ORFs) providing many potential proteins. These proteins may have important ...

Kinase Inhibitor Screening In Self-assembled Human Protein Microarrays

The screening of kinase inhibitors is crucial for better understanding properties of a drug and for the identification of potentially new targets with clinical implications. Several methodologies have been reported to accomplish such screening. However, each has its own limitations (e.g., the screening of only ATP analogues, restriction to using purified kinase domains, significant costs associated with testing more than a few kinases at a time, and lack of flexibility in screening protein kinases with novel mutations). Here, a new protocol that overcomes some of these limitations and can be used for the unbiased screening of kinase inhibitors is presented. A strength of this method is its ability to compare the activity of kinase inhibitors across multiple proteins, either between different kinases or different variants of the same kinase. Self-assembled protein microarrays generated through the expression of protein kinases by a human-based in vitro transcription and translation system (IVTT) are employed. The proteins displayed on the microarray are active, allowing for measurement of the effects of kinase inhibitors. The following procedure describes the protocol steps in detail, from the microarray generation and screening to the data analysis.

A detailed protocol for the generation of self-assembled human protein microarrays for the screening of kinase inhibitors is presented.

自己組み立てヒトタンパク質マイクロアレイにおけるキナーゼ阻害剤スクリーニング

The screening of kinase inhibitors is crucial for better understanding properties of a drug and for the identification of potentially new targets with ...

Quantification of Protein Interaction Network Dynamics using Multiplexed Co-Immunoprecipitation

Dynamic protein-protein interactions control cellular behavior, from motility to DNA replication to signal transduction. However, monitoring dynamic interactions among multiple proteins in a protein interaction network is technically difficult. Here, we present a protocol for Quantitative Multiplex Immunoprecipitation (QMI), which allows quantitative assessment of fold changes in protein interactions based on relative fluorescence measurements of Proteins in Shared Complexes detected by Exposed Surface epitopes (PiSCES). In QMI, protein complexes from cell lysates are immunoprecipitated onto microspheres, and then probed with a labeled antibody for a different protein in order to quantify the abundance of PiSCES. Immunoprecipitation antibodies are conjugated to different MagBead spectral regions, which allows a flow cytometer to differentiate multiple parallel immunoprecipitations and simultaneously quantify the amount of probe antibody associated with each. QMI does not require genetic tagging and can be performed using minimal biomaterial compared to other immunoprecipitation methods. QMI can be adapted for any defined group of interacting proteins, and has thus far been used to characterize signaling networks in T cells and neuronal glutamate synapses. Results have led to new hypothesis generation with potential diagnostic and therapeutic applications. This protocol includes instructions to perform QMI, from the initial antibody panel selection through to running assays and analyzing data. The initial assembly of a QMI assay involves screening antibodies to generate a panel, and empirically determining an appropriate lysis buffer. The subsequent reagent preparation includes covalently coupling immunoprecipitation antibodies to MagBeads, and biotinylating probe antibodies so they can be labeled by a streptavidin-conjugated fluorophore. To run the assay, lysate is mixed with MagBeads overnight, and then beads are divided and incubated with different probe antibodies, and then a fluorophore label, and read by flow cytometry. Two statistical tests are performed to identify PiSCES that differ significantly between experimental conditions, and results are visualized using heatmaps or node-edge diagrams.

Quantitative Multiplex Immunoprecipitation (QMI) uses flow cytometry for sensitive detection of differences in the abundance of targeted protein-protein interactions between two samples. QMI can be performed using a small amount of biomaterial, does not require genetically engineered tags, and can be adapted for any previously defined protein interaction network.

多重共免疫沈殿を用いたタンパク質相互作用ネットワークダイナミクスの定量化

Dynamic protein-protein interactions control cellular behavior, from motility to DNA replication to signal transduction. However, monitoring dynamic ...

ニトロセルロース結合アッセイを用いて細菌ヒスチジンキナーゼ自己リン酸化の定量化

要約

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この動画の章

共沈降によるF-アクチンへのタンパク質結合の測定

SDS ページを用いたリン酸 binding タグ LRRK2 活動によって Rab10 リン酸化検出

高効率巻き戻しと受容体リガンド結合領域の浄化法

小さい gtp アーゼプレニル化および分子量 GTP 結合を使用して膜分画と gtp アーゼ抗体法の検出

サイクリン依存性キナーゼ 1 の同定の in Vitroキナーゼによって特定のリン酸化のサイトの試金します。

散逸の Microgravimetry 結合タンパク質アネキシン A2 への水晶振動子を用いた固相担脂質膜リン脂質の結合動態を研究するには

多彩な生化学的アプローチを用いた新規 CK2 キナーゼ基板の識別

新しいキナーゼタンパク質の堅牢な生化学的アプローチを用いて分子レベルでの特性解析

自己組み立てヒトタンパク質マイクロアレイにおけるキナーゼ阻害剤スクリーニング

多重共免疫沈殿を用いたタンパク質相互作用ネットワークダイナミクスの定量化

ニトロセルロース結合アッセイを用いて細菌ヒスチジンキナーゼ自己リン酸化の定量化

要約

さらに動画を探す

この動画の章

共沈降によるF-アクチンへのタンパク質結合の測定

SDS ページを用いたリン酸 binding タグ LRRK2 活動によって Rab10 リン酸化検出

高効率巻き戻しと受容体リガンド結合領域の浄化法

小さい gtp アーゼ プレニル化および分子量 GTP 結合を使用して膜分画と gtp アーゼ抗体法の検出

サイクリン依存性キナーゼ 1 の同定の in Vitroキナーゼによって特定のリン酸化のサイトの試金します。

散逸の Microgravimetry 結合タンパク質アネキシン A2 への水晶振動子を用いた固相担脂質膜リン脂質の結合動態を研究するには

多彩な生化学的アプローチを用いた新規 CK2 キナーゼ基板の識別

新しいキナーゼタンパク質の堅牢な生化学的アプローチを用いて分子レベルでの特性解析

自己組み立てヒトタンパク質マイクロアレイにおけるキナーゼ阻害剤スクリーニング

多重共免疫沈殿を用いたタンパク質相互作用ネットワークダイナミクスの定量化

小さい gtp アーゼプレニル化および分子量 GTP 結合を使用して膜分画と gtp アーゼ抗体法の検出