Name: use of microscale thermophoresis to measure protein-lipid interactions
Uploaded: 2022-02-10T12:30:00
Description: Watch this Scientific Journal Video about Use of Microscale Thermophoresis to Measure Protein-Lipid Interactions at JoVE.com

この研究は、米国国立科学財団(MCB 1818310)からRAFへの助成金によって支援されました。この研究は、H. Lee Moffitt Cancer CenterのChemical Biology Core Facility/Protein Crystallography Unit(NIH/NCI:P30-CA076292)によって部分的に支援された。

1. 材料の準備
<ol><li>リン酸緩衝生理食塩水(PBS)を調製する:137 mM NaCl、2.5 mM KCl、10 mM NaH2PO4、および2 mM KH2PO4、pH 7.41。</li>
	<li>NTA-Atto 647 N染料を調製する。ストックNTA-Atto 647 N色素を100%DMSO溶液からトゥイーンなしのPBSに100nMに希釈する。</li>
	<li>Vam7-His8 – タンパク質を大腸菌 中の融合タンパク質として発現させ、Ni-NTAおよびサイズ排除<s...

<ol>
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A., 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=Label-free+microscale+thermophoresis+discriminates+sites+and+affinity+of+protein-ligand+binding.">Label-free microscale thermophoresis discriminates sites and affinity of protein-ligand binding.</a> Angewandte Chemie International Edition English. 51 (42), 10656-10659 (2012).</li><li>Starr, M. 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=Phosphatidic+acid+induces+conformational+changes+in+Sec18+protomers+that+prevent+SNARE+priming.">Phosphatidic acid induces conformational changes in Sec18 protomers that prevent SNARE priming.</a> Journal of Biological Chemistry. 294 (9), 3100-3116 (2019).</li><li>Miner, G. 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=The+Central+Polybasic+Region+of+the+Soluble+SNARE+(Soluble+N-Ethylmaleimide-sensitive+Factor+Attachment+Protein+Receptor)+Vam7+Affects+Binding+to+Phosphatidylinositol+3-Phosphate+by+the+PX+(Phox+Homology)+Domain.">The Central Polybasic Region of the Soluble SNARE (Soluble N-Ethylmaleimide-sensitive Factor Attachment Protein Receptor) Vam7 Affects Binding to Phosphatidylinositol 3-Phosphate by the PX (Phox Homology) Domain.</a> Journal of Biological Chemistry. 291 (34), 17651-17663 (2016).</li><li>Collins, M. D., Gordon, S. 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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stress+proteins:+virulence+factors+of+intracellular+disease+agents.">Stress proteins: virulence factors of intracellular disease agents.</a> Immunitat und Infektion. 17 (4), 124-128 (1989).</li><li>le Maire, M., Champeil, P., Moller, J. 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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+Microscale+Thermophoresis+to+Characterize+Hits+from+High-Throughput+Screening:+A+European+Lead+Factory+Perspective.">Using Microscale Thermophoresis to Characterize Hits from High-Throughput Screening: A European Lead Factory Perspective.</a> SLAS Discovery. 23 (3), 225-241 (2018).</li><li>Sparks, R. P., 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=A+small-molecule+competitive+inhibitor+of+phosphatidic+acid+binding+by+the+AAA++protein+NSF/Sec18+blocks+the+SNARE-priming+stage+of+vacuole+fusion.">A small-molecule competitive inhibitor of phosphatidic acid binding by the AAA+ protein NSF/Sec18 blocks the SNARE-priming stage of vacuole fusion.</a> Journal of Biological Chemistry. 294 (46), 17168-17185 (2019).</li></ol>

DiC8-PAへのVam7-His8結合の決定は、所与の相互作用に対して堅牢なフィッティングKDを提供し、これはPAリポソームに対するVam7-His8の測定されたKDよりもわずかに低い親和性である(未発表)。この差は、膜の欠如に起因する可能性が最も高く、これは一般に、膜特異的脂質結合相互作用に対する親和性の低下をもたらし、したがって、この相互作用に対す...

マイクロスケール熱泳動は、生化学的に関連するリガンド間の不会合定数(KD)および阻害定数(IC50)を決定する比較的新しい技術です。MSTの大手商業小売業者(NanoTemperなど)は、1)蛍光タグを必要とする標識フリーMSTと、2)各タンパク質に存在する芳香族残基の数に依存するタンパク質の固有の蛍光を使用した標識熱泳動の2つの一般的なMST技術を提供しています1。標識フリー熱泳動の欠点は、ほとんどの場合、タンパク質間相互作用の測定ができないことです。しかし、標識フリー熱泳動2に使用するために、トリプトファンなどの芳香族アミノ酸を含まないタンパク質を操作できる可能性があります。
MSTは、現在利用可能な技術で赤外線レーザーによって開始される微視的な温度場の誘導に応答して粒子の動きを測定します1。MSTは、十分なシグナル分離を生じさせることができる限り、タンパク質間相互作用、タンパク質-脂質相互作用、タンパク質-小分子間相互作用、競合実験、さらには小分子間の相互作用を測定するために使用することができる。さらに、MSTは、リポソームまたはナノディスクのいずれかに埋め込まれた膜 - タンパク質ベースの相互作用の測定を可能にする。標識サーモフォレーシスは、蛍光標識タグの使用を利用して、リガンドと分析物間のシグナルを化学的に制御可能な分離を可能にします。KD値は、低ナノモル濃度でのタンパク質結合を伴う相互作用の熱泳動を使用して得ることができ、ほとんどの場合、等温熱量測定(ITC)3に必要なものよりもはるかに低い濃度のタンパク質である。さらに、MSTには表面プラズモン共鳴(SPR)4に必要な厳密な緩衝要件がなく、標識熱泳動を使用して、遺伝子が挿入された蛍光タグ6を備えた完全に精製されていないタンパク質溶液5から目的のタンパク質の結合定数を測定することもできます。MSTの欠点は、SPR2のようにMSTの速度論的パラメータを容易に取得できないことである。
熱泳動測定は、溶液の局所的な温度差に依存します。この熱は赤外線レーザーから発生させることができる。MST装置は、赤外(IR)ビームに結合された蛍光検出器を有し、IRレーザーが標的とされる点における蛍光分子の局所的な濃度変化から蛍光の変化を拾うことができる。MSTデバイスは、溶液中で熱が発生するのと同じポイントに集束した蛍光検出器に直接結合されたIRターゲットレーザーを利用します。これにより、IRレーザーによって発生する熱の点での分子の枯渇に対応する温度変化の堅牢な検出が可能になります。測定された蛍光は、一般に、温度上昇に応答してIRレーザーに近づくほど減少する。結果として測定される差は、電荷、サイズ、溶媒和エントロピーなどの複数の要因に起因する可能性があります。これらの差は、熱の誘導または毛細血管の高温からより冷たい部分への分子の移動に応答した蛍光の変化として測定される。
キャピラリーに所定の溶液をロードするときは、キャピラリーの両端に空気を残し、キャピラリーを完全にいっぱいにロードしないことが重要です。市販の毛細血管は約10μLの溶液を保持する。溶液がキャピラリーの中心まで操作され、気泡(キャピラリーをロードする前に脱気する可能性がある)がなく、赤外線レーザーが標的とされるキャピラリーの中心から溶液を揺さぶらないようにラックを慎重にロードする限り、5μLの溶液で正確な測定を達成できます。レーザーが溶液と完全に接触しない場合、その結果は、その濃度に対して使用できない3つの出力のうちの1つになる可能性が最も高い:1)蛍光検出なしまたは低い、2)より高い蛍光検出(ギザギザのピークを有する可能性がある)、または3)所定の滴定からの他の値内の蛍光検出が、ギザギザで丸みを帯びていないピークを有する。
標識熱泳動では、200 を超える蛍光ユニットと 2000 未満の蛍光シグナルを持つのが最適です7。MSTデバイスは、0~100の範囲のLED強度を使用し、200以上または2000未満の信号を達成するために選択することができます。あるいは、異なる濃度の標識リガンドを使用して、蛍光シグナルを最適なレベルに修飾することができる。データ分析時に特定のMST測定値を基準としてキャップスキャンを実行することが重要です。キャップスキャンが不十分な場合、後で外れ値と判断される点が生じることがよくあります。キャップスキャンで単一のMST電力を測定する場合、各実行には約30分かかります。商用デバイスでは、MST 電力の変更が可能です。古いソフトウェアバージョンでは、これは0から100まで設定できました。それ以降のバージョンでは、低、中、または高のMSTを選択できます。堅牢なトレースを実現するには、研究者はこれらのそれぞれを試して、特定の相互作用に対して最も堅牢なデータが得られるMST設定を決定する必要があります。

<table>
	<tbody>
		<tr>
			<td>Cy5 Maleimide Mono-Reactive Dye</td>
			<td>GE Healthcare</td>
			<td>PA23031</td>
			<td>For protein labeleing</td>
		</tr>
		<tr>
			<td>Graphpad Prsim</td>
			<td>Graphpad software</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Monolith NT.115 Capillaries (1000 count)</td>
			<td>Nanotemper</td>
			<td>MO-K022</td>
			<td>Capillaries for MST</td>
		</tr>
		<tr>
			<td>Monolith NT.115 machine</td>
			<td>Nanotemper</td>
			<td></td>
			<td>University equipment</td>
		</tr>
		<tr>
			<td>NTA-Atto 647 N</td>
			<td>Sigma</td>
			<td>2175</td>
			<td>label for His tags</td>
		</tr>
		<tr>
			<td>Phosphatidylinositol 3-phosphate diC8 (PI(3)P diC8)</td>
			<td>Echelon</td>
			<td>P-3008</td>
			<td>Lipid for binding experiments</td>
		</tr>
	</tbody>
</table>

use of microscale thermophoresis to measure protein-lipid interactions

脂質とタンパク質との結合親和性を決定する能力は、膜トラフィッキング、シグナル伝達および細胞骨格リモデリングにおけるタンパク質間相互作用を理解する上で不可欠な部分である。このような相互作用を測定するための古典的なツールには、表面プラズモン共鳴(SPR)および等温滴定熱量測定(ITC)が含まれる。強力なツールではありますが、これらのアプローチには挫折があります。ITCは、脂質だけでなく、精製タンパク質を大量に必要とし、コストがかかり、製造が困難な場合があります。さらに、ITCとSPRは非常に時間がかかり、これらの実験を実行するコストを大幅に増加させる可能性があります。これらの制限を回避する方法の1つは、マイクロスケール熱泳動(MST)の比較的新しい技術を使用することです。MSTは、少量のサンプルを使用して、所与の結合事象の飽和曲線を得るために、高速かつ費用対効果が高い。現在、使用可能な MST システムには 2 つのタイプがあります。MSTの1つのタイプは、青色または赤色スペクトルの蛍光色素分子による標識を必要とする。第2のシステムは、UV範囲における芳香族アミノ酸の固有の蛍光に依存する。どちらのシステムも、赤外線レーザーからの局所的な熱誘導に応答して分子の動きを検出します。それぞれのアプローチには長所と短所があります。ラベルフリーMSTは、タグなしの天然タンパク質を使用することができる。しかし、医薬品を含む多くの分析物はUV範囲で蛍光を発し、正確なKD値の決定を妨げる可能性があります。対照的に、標識MSTは、UVとは対照的に可視範囲の測定可能な吸光度を有するリガンドに結合した蛍光標識プローブを利用して、測定可能なペアワイズ相互作用の多様性を可能にし、分析物からのシグナルを妨害する可能性を制限する。

これは、アフィニティー分析を使用したサンプル出力です。標識MSTを用いて、その天然基質の1つである可溶性ジオクタノイル(DiC8)PAに対するVam7-His8の結合定数を決定した9。図1は、50nMのVam7-His8に対して500μMから開始したDiC8 PAの1:1滴定の1つの試行からの熱泳動トレースを示しています。初期蛍光(赤外線レーザーがオンになる前の時間)、Tjump(赤外線レー...

Watch this Scientific Journal Video about Use of Microscale Thermophoresis to Measure Protein-Lipid Interactions at JoVE.com

Use of Microscale Thermophoresis to Measure Protein-Lipid Interactions

マイクロスケール熱泳動は、低い材料コストで結合定数を迅速に取得します。標識または標識フリーマイクロスケール熱泳動のいずれかが市販されている;しかしながら、標識フリーの熱泳動は、蛍光標識を用いて行うことができる多様な相互作用測定が可能ではない。当社は、標識熱泳動測定のためのプロトコルを提供しています。

タンパク質-脂質相互作用を測定するためのマイクロスケール熱泳動の使用

The ability to determine the binding affinity of lipids to proteins is an essential part of understanding protein-lipid interactions in membrane ...

標識マイクロスケール熱泳動は、多様な生化学的相互作用の解離定数(KD)を効率的に取得します。このプロトコルと添付のビデオは、酵母で唯一の可溶性SNAREタンパク質であるVam7とホスファチジン酸とのおおよその親和性を与える。これは、Vam7が他のタンパク質と相互作用する前に2つの膜と会合する方法である。この方法は、多様な生化学的相互作用のために、低コストで、高い再現性で、最小限のタンパク質コストでKDを迅速に取得する。この技術は、第1段階の医薬品開発に適しており、容易に入手できるKDが潜在的なリード化合物を決定することを可能にする。解析を開始する前に、MSTデバイス背面の電源スイッチをオンにし、制御ソフトウェアを開きます。コンピューターがデバイスに接続され、接続状態になっていることを確認し、[前 MST] を 3 秒、MST を 30 秒、蛍光回復を 1 秒後に設定します。各キャピラリーチューブについて、ターゲットリガンドの名前、リガンド分析物の名前、ターゲットの濃度、およびオートフィル滴定比を使用した最高滴定濃度を入力します。MST検出力の範囲を選択し、各検出力の値を入力して、最も堅牢な結合適合度をテストします。標識MSTサンプルを調製するには、Vam7-オクタヒスチジン溶液を200ナノモル濃度に希釈し、NTA-Atto 647色素を100ナノモル濃度に希釈します。Vam7-オクタヒスチジンとNTA-Atto 647色素を1:1の体積比で混合し、混合物を光から保護された室温で30分間座らせます。インキュベーションの最後に、色素とタンパク質の混合物を遠心分離し、使用前に最大数時間摂氏4度で溶液を保管します。その後、サンプルを準備し、染色されたリガンドを採取し、分析物を露出させる。サンプルのマイクロスケール熱泳動の場合は、デバイスを開き、キャピラリーラックをスライドさせて引き出します。サンプルキャピラリーをラックに積み込み、位置1で最も高い濃度で、制御ソフトウェアで赤色チャンネルを選択します。次に、ラックを装置にロードします。次に、MSTパワーの範囲を選択し、キャップスキャンMST測定を開始し、シフト応答を評価します。50ナノモルのNTA-Atto 647標識Vam7ドメインに対して500マイクロモルから始まるDiC8 PAの1:1滴定の1つの試験からの熱泳動痕跡をここに示します。初期蛍光、タイムジャンプ、および熱泳動を観察することができ、KDはこれらの測定値のいずれか1つまたは組み合わせから計算することができます。これらの結果から飽和曲線をプロットできます (たとえば、グラフに示すように、タイムジャンプ出力による熱フォレーシスの場合など)。一般に、MST結果は、対数スケールを用いて決定され、タンパク質濃度を考慮して、KDモデルを選択し、タンパク質濃度を入力することによって結合親和性を決定する。このプロトコルを実行するときは、溶液がキャピラリーの中央にあり、気泡がないこと、およびキャピラリーの外側にほこりや溶液がないことを確認してください。等温滴定熱量測定または表面プラズモン共鳴は、得られた実験KDを検証するために使用することができる。潜在的な協調バインディングがある場合、ITCは、リソースが与えられた場合、次に実行される論理的な方法になります。この技術により、従来の生物学研究者は、経路関係を決定するための研究に生物物理学的スクリーニング技術を組み込むことができました。この技術の一例は、標的タンパク質を内因性に発現させた細胞リセーゼであり、GFPタグは従来のプルダウンアッセイを実行するための新しい方法である。

use-of-microscale-thermophoresis-to-measure-protein-lipid-interactions

Research

JoVE Journal

Biochemistry

Mapping the Binding Site of an Aptamer on ATP Using MicroScale Thermophoresis

Characterization of molecular interactions in terms of basic binding parameters such as binding affinity, stoichiometry, and thermodynamics is an essential step in basic and applied science. MicroScale Thermophoresis (MST) is a sensitive biophysical method to obtain this important information. Relying on a physical effect called thermophoresis, which describes the movement of molecules through temperature gradients, this technology allows for the fast and precise determination of binding parameters in solution and allows the free choice of buffer conditions (from buffer to lysates/sera). MST uses the fact that an unbound molecule displays a different thermophoretic movement than a molecule that is in complex with a binding partner. The thermophoretic movement is altered in the moment of molecular interaction due to changes in size, charge, and hydration shell. By comparing the movement profiles of different molecular ratios of the two binding partners, quantitative information such as binding affinity (pM to mM) can be determined. Even challenging interactions between molecules of small sizes, such as aptamers and small compounds, can be studied by MST. Using the well-studied model interaction between the DH25.42 DNA aptamer and ATP, this manuscript provides a protocol to characterize aptamer-small molecule interactions. This study demonstrates that MST is highly sensitive and permits the mapping of the binding site of the 7.9 kDa DNA aptamer to the adenine of ATP.

MicroScale Thermophoresis (MST) is a sensitive technology to characterize aptamer-target interactions. This manuscript describes an MST protocol to characterize aptamer-small molecule interactions.

マイクロスケールの熱泳動を使用してATPのアプタマーの結合部位のマッピング

Characterization of molecular interactions in terms of basic binding parameters such as binding affinity, stoichiometry, and thermodynamics is an ...

生化学

Quantitative Immunofluorescence to Measure Global Localized Translation

The mechanisms regulating mRNA translation are involved in various biological processes, such as germ line development, cell differentiation, and organogenesis, as well as in multiple diseases. Numerous publications have convincingly shown that specific mechanisms tightly regulate mRNA translation. Increased interest in the translation-induced regulation of protein expression has led to the development of novel methods to study and follow de novo protein synthesis in cellulo. However, most of these methods are complex, making them costly and often limiting the number of mRNA targets that can be studied. This manuscript proposes a method that requires only basic reagents and a confocal fluorescence imaging system to measure and visualize the changes in mRNA translation that occur in any cell line under various conditions. This method was recently used to show localized translation in the subcellular structures of adherent cells over a short period of time, thus offering the possibility of visualizing de novo translation for a short period during a variety of biological processes or of validating changes in translational activity in response to specific stimuli.

This manuscript describes a method to visualize and quantify localized translation events in subcellular compartments. The approach proposed in this manuscript requires a basic confocal imaging system and reagents and is rapid and cost-effective.

グローバルを測定する定量的蛍光ローカライズ翻訳

The mechanisms regulating mRNA translation are involved in various biological processes, such as germ line development, cell differentiation, and ...

Biotinylated Cell-penetrating Peptides to Study Intracellular Protein-protein Interactions

Here we present a protocol to study intracellular protein-protein interactions that is based on the widely used biotin-avidin pull-down system. The modification presented includes the combination of this technique with cell-penetrating sequences. We propose to design cell-penetrating baits that can be incubated with living cells instead of cell lysates and therefore the interactions found will reflect those that occur within the intracellular context. Connexin43 (Cx43), a protein that forms gap junction channels and hemichannels is down-regulated in high-grade gliomas. The Cx43 region comprising amino acids 266-283 is responsible for the inhibition of the oncogenic activity of c-Src in glioma cells. Here we use TAT as the cell-penetrating sequence, biotin as the pull-down tag and the region of Cx43 comprised between amino acids 266-283 as the target to find intracellular interactions in the hard-to-transfect human glioma stem cells. One of the limitations of the proposed method is that the molecule used as bait could fail to fold properly and, consequently, the interactions found could not be associated with the effect. However, this method can be especially interesting for the interactions involved in signal transduction pathways because they are usually carried out by intrinsically disordered regions and, therefore, they do not require an ordered folding. In addition, one of the advantages of the proposed method is that the relevance of each residue on the interaction can be easily studied. This is a modular system; therefore, other cell-penetrating sequences, other tags, and other intracellular targets can be employed. Finally, the scope of this protocol is far beyond protein-protein interaction because this system can be applied to other bioactive cargoes such as RNA sequences, nanoparticles, viruses or any molecule that can be transduced with cell-penetrating sequences and fused to pull-down tags to study their intracellular mechanism of action.

This is a protocol to study intracellular protein-protein interactions based on the biotin-avidin pull-down system with the novelty of combining cell-penetrating sequences. The main advantage is that the target sequence is incubated with living cells instead of cell lysates and therefore the interactions will occur within the cellular context.

細胞内タンパク質間相互作用を研究するビオチン化セル透過ペプチド

Here we present a protocol to study intracellular protein-protein interactions that is based on the widely used biotin-avidin pull-down system. The ...

Measuring Interactions of Globular and Filamentous Proteins by Nuclear Magnetic Resonance Spectroscopy (NMR) and Microscale Thermophoresis (MST)

Filamentous proteins such as vimentin provide organization within cells by providing a structural scaffold with sites that bind proteins containing plakin repeats. Here, a protocol for detecting and measuring such interactions is described using the globular plakin repeat domain of envoplakin and the helical coil of vimentin. This provides a basis for determining whether a protein binds vimentin (or similar filamentous proteins) and for measurement of the affinity of the interaction. The globular protein of interest is labeled with 15N and titrated with vimentin protein in solution. A two-dimensional NMR spectrum is acquired to detect interactions by observing changes in peak shape or chemical shifts, and to elucidate effects of solution conditions including salt levels, which influence vimentin quaternary structure. If the protein of interest binds the filamentous ligand, the binding interaction is quantified by MST using the purified proteins. The approach is a straightforward way for determining whether a protein of interest binds a filament, and for assessing how alterations, such as mutations or solution conditions, affect the interaction.

Measuring Interactions of Globular and Filamentous Proteins by Nuclear Magnetic Resonance Spectroscopy NMR and Microscale Thermophoresis MST

Here, we present a protocol for the production and purification of proteins that are labeled with stable isotopes, and subsequent characterization of protein-protein interactions using Nuclear Magnetic Resonance (NMR) spectroscopy and MicroScale Thermophoresis (MST) experiments.

核磁気共鳴分光法 (NMR) とマイクロ働く (MST) による球状と糸状のタンパク質の相互作用を測定

Filamentous proteins such as vimentin provide organization within cells by providing a structural scaffold with sites that bind proteins containing plakin ...

Atomic Absorbance Spectroscopy to Measure Intracellular Zinc Pools in Mammalian Cells

Transition metals are essential micronutrients for organisms but can be toxic to cells at high concentrations by competing with physiological metals in proteins and generating redox stress. Pathological conditions that lead to metal depletion or accumulation are causal agents of different human diseases. Some examples include anemia, acrodermatitis enteropathica, and Wilson&#8217;s and Menkes&#8217; diseases. It is therefore important to be able to measure the levels and transport of transition metals in biological samples with high sensitivity and accuracy in order to facilitate research exploring how these elements contribute to normal physiological functions and toxicity. Zinc (Zn), for example, is a cofactor in many mammalian proteins, participates in signaling events, and is a secondary messenger in cells. In excess, Zn is toxic and can inhibit absorption of other metals, while in deficit, it can lead to a variety of potentially lethal conditions.

Graphite furnace atomic absorption spectroscopy (GF-AAS) provides a highly sensitive and effective method for determining Zn and other transition metal concentrations in diverse biological samples. Electrothermal atomization via GF-AAS quantifies metals by atomizing small volumes of samples for subsequent selective absorption analysis using wavelength of excitation of the element of interest. Within the limits of linearity of the Beer-Lambert Law, the absorbance of light by the metal is directly proportional to concentration of the analyte. Compared to other methods of determining Zn content, GF-AAS detects both free and complexed Zn in proteins and possibly in small intracellular molecules with high sensitivity in small sample volumes. Moreover, GF-AAS is also more readily accessible than inductively coupled plasma mass spectrometry (ICP-MS) or synchrotron-based X-ray fluorescence. In this method, the systematic sample preparation of different cultured cell lines for analyses in a GF-AAS is described. Variations in this trace element were compared in both whole cell lysates and subcellular fractions of proliferating and differentiated cells as proof of principle.

Cultured primary or established cell lines are commonly used to address fundamental biological and mechanistic questions as an initial approach before using animal models. This protocol describes how to prepare whole cell extracts and subcellular fractions for studies of zinc (Zn) and other trace elements with atomic absorbance spectroscopy.

哺乳類細胞内の細胞内亜鉛プールを測定するための原子吸光度分光法

Transition metals are essential micronutrients for organisms but can be toxic to cells at high concentrations by competing with physiological metals in ...

Use of Alu Element Containing Minigenes to Analyze Circular RNAs

In addition to linear mRNAs, many eukaryotic genes generate circular RNAs. Most circular RNAs are generated by joining a 5&#39; splice site with an upstream 3&#39; splice site within a pre-mRNA, a process called back-splicing. This circularization is likely aided by secondary structures in the pre-mRNA that bring the splice sites into close proximity. In human genes, Alu elements are thought to promote these secondary RNA structures, as Alu elements are abundant and exhibit base complementarities with each other when present in opposite directions in the pre-mRNA. Here, we describe the generation and analysis of large, Alu element containing reporter genes that form circular RNAs. Through optimization of cloning protocols, reporter genes with up to 20 kb insert length can be generated. Their analysis in co-transfection experiments allows the identification of regulatory factors. Thus, this method can identify RNA sequences and cellular components involved in circular RNA formation.

Use of Alu Element Containing Minigenes to Analyze Circular RNAs

We clone and analyze reporter genes generating circular RNAs. These reporter genes are larger than constructs to analyze linear splicing and contain Alu elements. To investigate the circular RNAs, the constructs are transfected into cells and resulting RNA is analyzed using RT-PCR after removal of linear RNA.

円形RNA解析に対する小遺伝子を含むAlu要素の使用

In addition to linear mRNAs, many eukaryotic genes generate circular RNAs. Most circular RNAs are generated by joining a 5&#39; splice site with an ...

Nonradioactive Assay to Measure Polynucleotide Phosphorylation of Small Nucleotide Substrates

Polynucleotide kinases (PNKs) are enzymes that catalyze the phosphorylation of the 5&#39; hydroxyl end of DNA and RNA oligonucleotides. The activity of PNKs can be quantified using direct or indirect approaches. Presented here is a direct, in vitro approach to measure PNK activity that relies on a fluorescently-labeled oligonucleotide substrate and polyacrylamide gel electrophoresis. This approach provides resolution of the phosphorylated products while avoiding the use of radiolabeled substrates. The protocol details how to set up the phosphorylation reaction, prepare and run large polyacrylamide gels, and quantify the reaction products. The most technically challenging part of this assay is pouring and running the large polyacrylamide gels; thus, important details to overcome common difficulties are provided. This protocol was optimized for Grc3, a PNK that assembles into an obligate pre-ribosomal RNA processing complex with its binding partner, the Las1 nuclease. However, this protocol can be adapted to measure the activity of other PNK enzymes. Moreover, this assay can also be modified to determine the effects of different components of the reaction, such as the nucleoside triphosphate, metal ions, and oligonucleotides.

This protocol describes a nonradioactive assay to measure kinase activity of polynucleotide kinases (PNKs) on small DNA and RNA substrates.

小ヌクレオチド基質のポリヌクレオチドリン酸化を測定する非放射性アッセイ

Polynucleotide kinases (PNKs) are enzymes that catalyze the phosphorylation of the 5&#39; hydroxyl end of DNA and RNA oligonucleotides. The activity of ...

CD Spectroscopy to Study DNA-Protein Interactions

Circular dichroism (CD) spectroscopy is a simple and convenient method to investigate the secondary structure and interactions of biomolecules. Recent advancements in CD spectroscopy have enabled the study of DNA-protein interactions and conformational dynamics of DNA in different microenvironments in detail for a better understanding of transcriptional regulation in vivo. The area around a potential transcription zone needs to be unwound for transcription to occur. This is a complex process requiring the coordination of histone modifications, binding of the transcription factor to DNA, and other chromatin remodeling activities. Using CD spectroscopy, it is possible to study conformational changes in the promoter region caused by regulatory proteins, such as ATP-dependent chromatin remodelers, to promote transcription. The conformational changes occurring in the protein can also be monitored. In addition, queries regarding the affinity of the protein towards its target DNA and sequence specificity can be addressed by incorporating mutations in the target DNA. In short, the unique understanding of this sensitive and inexpensive method can predict changes in chromatin dynamics, thereby improving the understanding of transcriptional regulation.

The interaction of an ATP-dependent chromatin remodeler with a DNA ligand is described using CD spectroscopy. The induced conformational changes on a gene promoter analyzed by the peaks generated can be used to understand the mechanism of transcriptional regulation.

DNA-タンパク質相互作用を研究するためのCD分光法

Circular dichroism (CD) spectroscopy is a simple and convenient method to investigate the secondary structure and interactions of biomolecules. Recent ...

Concanavalin A-Based Sedimentation Assay to Measure Substrate Binding of Glucan Phosphatases

Glucan phosphatases belong to the larger family of dual specificity phosphatases (DSP) that dephosphorylate glucan substrates, such as glycogen in animals and starch in plants. The crystal structures of glucan phosphatase with model glucan substrates reveal distinct glucan-binding interfaces made of DSP and carbohydrate-binding domains. However, quantitative measurements of glucan-glucan phosphatase interactions with physiologically relevant substrates are fundamental to the biological understanding of the glucan phosphatase family of enzymes and the regulation of energy metabolism. This manuscript reports a Concanavalin A (ConA)-based in vitro sedimentation assay designed to detect the substrate binding affinity of glucan phosphatases against different glucan substrates.&#160;As a proof of concept, the dissociation constant (KD) of glucan phosphatase Arabidopsis thaliana Starch Excess4 (SEX4) and amylopectin was determined. The characterization of SEX4 mutants and other members of the glucan phosphatase family of enzymes further demonstrates the utility of this assay to assess the differential binding of protein- carbohydrate interactions. These data demonstrate the suitability of this assay to characterize a wide range of starch and glycogen interacting proteins.

This method describes a lectin-based in vitro sedimentation assay to quantify the binding affinity of glucan phosphatase and amylopectin. This co-sedimentation assay is reliable for measuring glucan phosphatase substrate binding and can be applied to various solubilized glucan substrates.

グルカンホスファターゼの基質結合を測定するためのコンカナバリンAベースの沈降アッセイ

Glucan phosphatases belong to the larger family of dual specificity phosphatases (DSP) that dephosphorylate glucan substrates, such as glycogen in animals ...

安定同位体トレーシングと LC-MS 分析による DHODH および SDH 活性の測定

Oxygen-Independent Assays to Measure Mitochondrial Function in Mammals

The flow of electrons in the mitochondrial electron transport chain (ETC) supports multifaceted biosynthetic, bioenergetic, and signaling functions in mammalian cells. As oxygen (O2) is the most ubiquitous terminal electron acceptor for the mammalian ETC, the O2 consumption rate is frequently used as a proxy for mitochondrial function. However, emerging research demonstrates that this parameter is not always indicative of mitochondrial function, as fumarate can be employed as an alternative electron acceptor to sustain mitochondrial functions in hypoxia. This article compiles a series of protocols that allow researchers to measure mitochondrial function independently of the O2 consumption rate. These assays are particularly useful when studying mitochondrial function in hypoxic environments. Specifically, we describe methods to measure mitochondrial ATP production, de novo pyrimidine biosynthesis, NADH oxidation by complex I, and superoxide production. In combination with classical respirometry experiments, these orthogonal and economical assays will provide researchers with a more comprehensive assessment of mitochondrial function in their system of interest.

著者スポットライト:哺乳類のミトコンドリア機能を測定するための酸素に依存しないアッセイ  

Here, we present a compilation of assays to directly measure mitochondrial function in mammalian cells independently of their ability to consume molecular oxygen.

哺乳類のミトコンドリア機能を測定するための酸素非依存性アッセイ(英語)

The flow of electrons in the mitochondrial electron transport chain (ETC) supports multifaceted biosynthetic, bioenergetic, and signaling functions in ...