アッセイを開始する前に、我々は、以下の試薬が正しく準備されていることを確認する必要があります。 <ol><li> 50mMリン酸カリウム緩衝液、pH7.5の。カリウムphospate二塩基、水を精製し、1M HClでpHを調整するの三水和物11.4 mg / mlのを使用して準備します。この溶液を37で使用する° Cの前に配置されます。 </li><li> 50mMリン酸カリウム緩衝液中でカゼインの6.5 mg / mlに混合して調製した0.65％重量/容積カゼイン溶液、。均一な分散が達成されるまで徐々に緩やかに攪拌しながら溶液の温度は℃で約10分間80から85に増​​加した。それは解決策を沸騰しないように非常に重要です。 NaOHとHClで必要に応じてpHを調整されます。 </li><li>精製水を6.1Nの株式1時55分を希釈した110 mMのトリクロロ酢酸溶液を、。トリクロロ酢酸は強酸であり、取り扱いには注意してください。 </li><li> 0.5mMのフォリン＆Ciolcalteaの、または直接プロテアーゼの活性に関連する測定可能な色の変化を生成するチロシンと反応するソリューションですフォリンのフェノール試薬、。フォリンのフェノール試薬は酸であり、取り扱いには注意してください。 </li><li>精製水にanyhydrous炭酸ナトリウム53 mg / mlのを用いて調製した500mMの炭酸ナトリウム溶液、。 37 5mMの酢酸カルシウムと10mMの酢酸ナトリウム緩衝液、pH7.5、で構成される酵素希釈液を、℃のこのソリューションは、我々は固体プロテアーゼのサンプルを溶解または酵素液を希釈するために使用するものです。 </li><li> 1.1 mmのL -チロシン標準原液。精製水に0.2 mg / mlのL -チロシンを用いて調製し、チロシンが溶けるまで穏やかに加熱。カゼインと同様に、このソリューションを沸騰されません。 L -チロシン標準を室温に戻します。このソリューションは、当社の標準曲線を作成するためにさらに希釈されます。 </li><li>プロテアーゼ溶液。使用する直前に、ステップ6で準備酵素希釈液にプロテアーゼを溶解する。 </li></ol>必要に応じて、0.1〜0.2単位/ mlに酵素希釈液を用いて溶解されている所定の活動の固体プロテアーゼのサンプルを、使用してください。このソリューションは、品質管理アッセイの陽性コントロールとして、我々は酵素活性を決定するために実行する計算のための検証として提供しています。 プロテアーゼアッセイや標準曲線を設定する<ol><li>このアッセイを開始するには、約15 MLSを保持する適切なバイアルを見つける。テストされる各酵素について、4つのバイアルが必要です。 1バイアルは、空白として使用され、そして3人は、プロテアーゼの三希釈液のアッセイの活動に使用されます。お互いに最​​終計算をチェックする際に三希釈に便利です。 <ol><li> fourバイアルの各セットに、私たちの0.65％カゼイン溶液の5mlsを追加。それらを37℃の水浴中で平衡化させて° Cを約5分間。 </li><li>酵素の試験のサンプルバイアルの3つにテストされるソリューションではなく、ブランクの様々なボリュームを追加します。ちょうど10分間37 ° Cのために渦巻くとインキュベートして混合。このインキュベーション時間中のプロテアーゼ活性およびチロシンの必然的な解放は、測定および試験サンプル間で比較されるものです。 </li></ol></li><li>この10分間のインキュベーション後、反応を停止するには、各チューブにTCA試薬の5mlのを追加します。次に、各チューブ内の酵素溶液の最終体積が1ミリリットルになるように、、各チューブにさえブランクを酵素溶液の適切な量を追加します。これは、酵素自体の吸光度値を考慮すると、各チューブに最終容量が等しいかどうかを確認するために行われます。 37ソリューションをインキュベート℃で30分間。 この30分間のインキュベーションの間に、あなたのチロシン標準希釈液をセットアップすることもできます。簡単に8 MLSを保持できる6 DRAMバイアル（ドラムのバイアルは、ポリプロピレンチューブに置換することができます）を使用してください。 0.05、0.10、0.20、0.40、0.50シックスバイアルに、MLSに以下のボリュームで1.1 mmのチロシン標準のストック溶液を加える。空白に任意のチロシン標準を追加しないでください。下の規格は、ほとんど色の変化が得られる不純なテストサンプル用に必要になる場合があります。チロシン標準溶液を添加した後は、2 MLSのボリュームをもたらすための基準の各々に精製水の適切な量を追加します。 </li><li> 30分間のインキュベーションの後、試験溶液と0.45ミクロンポリエーテルスルホンシリンジフィルターを使用して空白の各フィルタをかける。ろ過は、サンプルから任意の不溶物を除去するために必要です。 <ol><li>少なくとも8 MLSを保持できる4ドラムバイアルにろ過2テストサンプルのMLSと空白のろ液を追加。規格が準備されたバイアルの同じタイプを使用することができます。 </li><li>規格と標準空白を含むバイアルのすべてに、炭酸ナトリウムの5mlsを追加。最良の結果を得るには、すぐ後にフォリン試薬1mlを加える。 </li><li>フォリン試薬の添加によって作成された任意のpHの低下を調整するために炭酸ナトリウムを追加。 </li><li>テストサンプルとTESに炭酸ナトリウムを追加tのブランク。これらのソリューションは、炭酸ナトリウムの添加後に濁ってくる。無料のチロシンと主に反応するフォリン試薬を、追加。 </li><li>渦巻きによってDRAMのバイアルを混合し、30分間37℃でインキュベートする。 </li></ol>このインキュベーション後に、基準がチロシン付加量と相関する色のグラデーションを持っていることに気付くはずです。チロシンの最高濃度は、最も濃い色に現れる。また、私たちのテストサンプルでかなりの色の変化に気づくことができる。これらのソリューションの2mlsは、適切なキュベットに0.45ミクロンポリエーテルスルホンシリンジフィルターを使用してフィルタリングされます。アッセイが行われるので、今度は私たちの吸光度値を記録するために分光光度計に進むことができます。 </li></ol>吸光度を測定し、酵素活性の計算<ol><li>サンプルの吸光度は、660nmの波長を用いて分光光度計によって測定されます。光パスは、1センチメートルに設定されています。規格、標準的な空白、別のテストサンプル、およびテストのブランクの吸光度値を記録します。すべてのデータが収集された後、標準曲線を作成することができます。曲線を生成するために、標準と標準のブランクの間の吸光度の差を計算する必要があります。これは、標準溶液中のチロシンの量に起因する吸光度の値です。この簡単な計算の後、Y軸上の私たちの基準の吸光度の変化に対し、X軸上の私たちの5標準の各マイクロモルの量をプロットするグラフプログラムを使用して標準曲線を作成します。 <table style="width: 416px; height: 118px;" border="0"><tbody><tr><th style="text-align:center">チロシン標準の音量</th><th style="text-align:center">チロシンuMoles </th></tr><tr><td style="text-align:center"> 0.05 </td><td style="text-align:center"> 0.055 </td></tr><tr><td style="text-align:center"> 0.10 </td><td style="text-align:center"> 0.111 </td></tr><tr><td style="text-align:center"> 0.20 </td><td style="text-align:center"> 0.221 </td></tr><tr><td style="text-align:center"> 0.40 </td><td style="text-align:center"> 0.442 </td></tr><tr><td style="text-align:center"> 0.50 </td><td style="text-align:center"> 0.553 </td></tr></tbody></table></li><li>データ点を入力したら、最高のフィット感と、対応するスロープ式のラインを生成する。 試験サンプルの吸光度とテストのブランクの吸光度との差を計算することにより、テストサンプルの吸光度の変化を見つける。スロープ式にテストサンプルの1つのための吸光度値を挿入し、解決することは、この特定のタンパク質分解反応中のチロシン解放のマイクロモルになります。 / mlあたり単位で酵素の活性を得るために、次の計算を実行します。 （umoleチロシン同等物が放出さ）×（11） 単位/ mlの酵素= __________________________________________ （1）×（10）×（2） アッセイの11 =総体積（ミリリットル） アッセイの10 =時間（分単位）単位の定義に従って 1酵素の酵素の=ボリューム（ミリリットル）を使用 比色定量に使用されている2 =ボリューム（ミリリットル） 斜面の方程式から得られるマイクロモルのチロシンに相当リリースの数を取り、我々の場合に11mlsであるMLS、のアッセイの合計量を掛けます。他の3つの量がこの値を分ける：（のは1ミリリットルを使用できるように）変化させた我々は10分間実行したアッセイの時間、、アッセイで使用される酵素の量、、比色検出に使用されるミリリットルの容積、これかもしれないあなたのキュベットに基づいて異なります。私たちは2 MLSを使用する。 </li><li> &quot;ユニット&quot;と呼ばれるプロテアーゼ活性の分収率の測定に時間で割ったチロシンのマイクロモル。私たちは、単位/ mlの条件で酵素活性のmeasurmentを残して、分子と分母の体積測定の単位を取り消すことができます。酵素希釈液で希釈した固体プロテアーゼのサンプルでのアクティビティを決定するために、我々はmg / mlで、もともとこのアッセイで使用される固体の濃度で単位/ mlで私たちの活動を割ります。、単位/ mgの面での活動を私たちに残して。 ユニット/ ml酵素 単位/ mgの固体= _____________________ MGソリッド/ mlの酵素 </li></ol>

<ol>
	<li>Anson, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> J. Gen. Physiol. 22, 79-89 (1938).</li><li>Folin, O., Ciocalteau, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> J. Biol. Chem. 73, 627 (1929).</li></ol>

私達はちょうどシグマのユニバーサルプロテアーゼ活性のアッセイを用いてプロテアーゼ活性を分析する方法を示してきました。さらに、このアッセイでは、実験のためにそれらを受け取る前に、私たちのプロテアーゼが正確に活動を決定していることを確認するのに便利です。これまで見てきたように、この手順を実行するとき、それは徐々にではなく、それらを沸かすためにカゼインおよびチロシンソリューションの両方を加熱することを忘れないように極めて重要です。また、それは両方の規格のために、あなたが持っている各テストサンプルのための別のブランクを準備することが重要です。

<table>
	<tbody>
		<tr>
			<td>Protease</td>
			<td>Sigma-Aldrich</td>
			<td>P4630</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Phosphate, Dibasic, Trihydrate</td>
			<td>Sigma-Aldrich</td>
			<td>P5504</td>
			<td></td>
		</tr>
		<tr>
			<td>Casein</td>
			<td>Sigma-Aldrich</td>
			<td>C7078</td>
			<td></td>
		</tr>
		<tr>
			<td>Trichloroacetic Acid</td>
			<td>Sigma-Aldrich</td>
			<td>T0699</td>
			<td></td>
		</tr>
		<tr>
			<td>Folin &#38; Ciocalteu&#39;s Phenol Reagent</td>
			<td>Sigma-Aldrich</td>
			<td>F9252</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Carbonate, Anhydrous</td>
			<td>Sigma-Aldrich</td>
			<td>S2127</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Acetate, Trihydrate</td>
			<td>Sigma-Aldrich</td>
			<td>S8625</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Acetate</td>
			<td>Sigma-Aldrich</td>
			<td>C1000</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Tyrosine, Free Base</td>
			<td>Sigma-Aldrich</td>
			<td>T3754</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease Profiler Kit</td>
			<td>Sigma-Aldrich</td>
			<td>PP0500</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease Fluorescent Detection Kit</td>
			<td>Sigma-Aldrich</td>
			<td>PF0100</td>
			<td></td>
		</tr>
	</tbody>
</table>

sigma's non-specific protease activity assay - casein as a substrate

プロテアーゼはペプチド結合を切断。ラボでは、それはしばしば測定および/またはプロテアーゼの活性を比較する必要があります。シグマの非特異的プロテアーゼ活性のアッセイは、我々は品質管理の手順の中でやっているプロテアーゼの活性を、決定するために標準化された手順として使用できます。このアッセイでは、カゼインが基質として機能します。我々がテストしているプロテアーゼはカゼインを消化するときに、アミノ酸のチロシンが他のアミノ酸とペプチドフラグメントと一緒に遊離させる。フォリンとCiocalteusフェノール、またはフォリン試薬は、主に定量化し、分光光度計で吸光度値として測定される青色の発色団を、生成する無料のチロシンと反応する。カゼインから解放されより多くのチロシン、より多くの発色団が生成され、プロテアーゼの活性を強く。プロテアーゼの活性によって生成された吸光度はマイクロモルのチロシンの量と吸光度の変化を関連させるFC試薬とチロシンの既知量を反応させることによって生成された標準曲線と比較されています。標準曲線からタンパク質分解酵素サンプルの活性は、毎分カゼインから遊離チロシン同等物のマイクロモルの量である、ユニットの観点から決定することができます。 中国語のこの記事を表示するには、 <a href="http://jove.com/index/details.stp?ID=910">ここをクリック</a>

Watch this Scientific Journal Video about Sigma's Non-specific Protease Activity Assay - Casein as a Substrate at JoVE.com

Sigma&#039;s Non-specific Protease Activity Assay - Casein as a Substrate

プロテアーゼはペプチド結合を切断。ラボでは、それはしばしば測定および/またはプロテアーゼの活性を比較する必要があります。シグマの非特異的プロテアーゼ活性のアッセイは、プロテアーゼの活性を決定するために標準化された手順として使用できます。

シグマの非特異的プロテアーゼの活性アッセイ - 基質としてカゼイン

Proteases break peptide bonds. In the lab, it is often necessary to measure and/or compare the activity of proteases.  Sigma's non-specific protease ...

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Sigma's Non-specific Protease Activity Assay - Casein as a Substrate

Unit 1

Alcohols and Phenols

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76.0K ビュー.  Sigma Aldrich.  ChineseVersion 

ビデオ: Sigma's Non-specific Protease Activity Assay - Casein as a Substrate

Rapid Genotyping of Mouse Tissue Using Sigma's Extract-N-Amp Tissue PCR Kit

Genomic detection of DNA via PCR amplification and detection on an electrophoretic gel is a standard way that the genotype of a tissue sample is determined. Conventional preparation of tissues for PCR-ready DNA often take several hours to days, depending on the tissue sample. The genotype of the sample may thus be delayed for several days, which is not an option for many different types of experiments. Here we demonstrate the complete genotyping of a mouse tail sample, including tissue digestion and PCR readout, in one and a half hours using Sigma's SYBR Green Extract-N-Amp Tissue PCR Kit. First, we demonstrate the fifteen-minute extraction of DNA from the tissue sample. Then, we demonstrate the real time read-out of the PCR amplification of the sample, which allows for the identification of a positive sample as it is being amplified. Together, the rapid extraction and real-time readout allow for a prompt identification of genotype of a variety different types of tissues through the reliable method of PCR.

Rapid Genotyping of Mouse Tissue Using Sigma&#039;s Extract-N-Amp Tissue PCR Kit

The complete genotyping of a mouse tail sample, including tissue digestion and PCR readout, is done in one and a half hours using Sigma's SYBR Green Extract-N-Amp Tissue PCR kit.

シグマのエキス- N - Ampの組織PCRキットを使用してマウス組織の急速なジェノタイピング

Genomic detection of DNA via PCR amplification and detection on an electrophoretic gel is a standard way that the genotype of a tissue sample is ...

生物学

Use of the Protease Fluorescent Detection Kit to Determine Protease Activity

The Protease Fluorescent Detection Kit provides ready-to-use reagents for detecting the presence of protease activity. This simple assay to detect protease activity uses casein labeled with fluorescein isothiocyanate (FITC) as the substrate.
Protease activity results in the cleavage of the FITC-labeled casein substrate into smaller fragments, which do not precipitate under acidic conditions. After incubation of the protease sample and substrate, the reaction is acidified with the addition of trichloroacetic acid (TCA). The mixture is then centrifuged with the undigested substrate forming a pellet and the smaller, acid soluble fragments remaining in solution. The supernatant is neutralized and the fluorescence of the FITC-labeled fragments is measured.
The described kit procedure detects the trypsin protease control at a concentration of approximately 0.5 &mu;g/ml (5 ng of trypsin added to the assay). This sensitivity can be increased with a longer incubation time, up to 24 hours. The assay is performed in microcentrifuge tubes and procedures are provided for fluorescence detection using either cuvettes or multiwell plates.

The Protease Fluorescent Detection Kit is designed for the measurement of protease activity using fluorometry. It is also suitable for detection of trace amounts of protease contamination. The method is based on the proteolytic hydroysis of a proprietary formulation of a FITC-labeled casein substrate.

プロテアーゼ活性を測定するためにプロテアーゼ蛍光検出キットの使用

The Protease Fluorescent Detection Kit provides ready-to-use reagents for detecting the presence of protease activity. This simple assay to detect ...

An Assay for Measuring the Activity of Escherichia coli Inducible Lysine Decarboxyase

Escherichia coli is an enteric bacterium that is capable of growing over a wide range of pH values (pH 5 - 9)1 and, incredibly, is able to survive extreme acid stresses including passage through the mammalian stomach where the pH can fall to as low as pH 1 - 22. To enable such a broad range of acidic pH survival, E. coli possesses four different inducible amino acid decarboxylases that decarboxylate their substrate amino acids in a proton-dependent manner thus raising the internal pH. The decarboxylases include the glutamic acid decarboxylases GadA and GadB3, the arginine decarboxylase AdiA4, the lysine decarboxylase LdcI5, 6 and the ornithine decarboxylase SpeF7. All of these enzymes utilize pyridoxal-5'-phospate as a co-factor8 and function together with inner-membrane substrate-product antiporters that remove decarboxylation products to the external medium in exchange for fresh substrate2. In the case of LdcI, the lysine-cadaverine antiporter is called CadB. Recently, we determined the X-ray crystal structure of LdcI to 2.0 &#197;, and we discovered a novel small-molecule bound to LdcI the stringent response regulator guanosine 5'-diphosphate,3'-diphosphate (ppGpp) 14. The stringent response occurs when exponentially growing cells experience nutrient deprivation or one of a number of other stresses9. As a result, cells produce ppGpp which leads to a signaling cascade culminating in the shift from exponential growth to stationary phase growth10. We have demonstrated that ppGpp is a specific inhibitor of LdcI 14. Here we describe the lysine decarboxylase assay, modified from the assay developed by Phan et al.11, that we have used to determine the activity of LdcI and the effect of pppGpp/ppGpp on that activity. The LdcI decarboxylation reaction removes the &alpha;-carboxy group of L-lysine and produces carbon dioxide and the polyamine cadaverine (1,5-diaminopentane)5. L-lysine and cadaverine can be reacted with 2,4,6-trinitrobenzensulfonic acid (TNBS) at high pH to generate N,N'-bistrinitrophenylcadaverine (TNP-cadaverine) and N,N&#8242;-bistrinitrophenyllysine (TNP-lysine), respectively11. The TNP-cadaverine can be separated from the TNP-lysine as the former is soluble in organic solvents such as toluene while the latter is not (See Figure 1). The linear range of the assay was determined empirically using purified cadaverine.

An Assay for Measuring the Activity of Escherichia coli Inducible Lysine Decarboxyase

The activity of the inducible lysine decarboxylase is monitored by reacting the substrate L-lysine and the product cadaverine with 2,4,6-trinitrobenzensulfonic acid to form adducts that have differential solubility in toluene.

の活性を測定するアッセイ大腸菌誘導性リジンDecarboxyase

Escherichia coli is an enteric bacterium that is capable of growing over a wide range of pH values (pH 5 - 9)1 and, incredibly, is able to survive extreme ...

A Matrigel-Based Tube Formation Assay to Assess the Vasculogenic Activity of Tumor Cells

Over the past several decades, a tube formation assay using growth factor-reduced Matrigel has been typically employed to demonstrate the angiogenic activity of vascular endothelial cells in vitro1-5. However, recently growing evidence has shown that this assay is not limited to test vascular behavior for endothelial cells. Instead, it also has been used to test the ability of a number of tumor cells to develop a vascular phenotype6-8. This capability was consistent with their vasculogenic behavior identified in xenotransplanted animals, a process known as vasculogenic mimicry (VM)9. There is a multitude of evidence demonstrating that tumor cell-mediated VM plays a vital role in the tumor development, independent of endothelial cell angiogenesis6, 10-13. For example, tumor cells were found to participate in the blood perfused, vascular channel formation in tissue samples from melanoma and glioblastoma patients8, 10, 11. Here, we described this tubular network assay as a useful tool in evaluation of vasculogenic activity of tumor cells. We found that some tumor cell lines such as melanoma B16F1 cells, glioblastoma U87 cells, and breast cancer MDA-MB-435 cells are able to form vascular tubules; but some do not such as colon cancer HCT116 cells. Furthermore, this vascular phenotype is dependent on cell numbers plated on the Matrigel. Therefore, this assay may serve as powerful utility to screen the vascular potential of a variety of cell types including vascular cells, tumor cells as well as other cells.

A tube formation assay is used to evaluate vascular activity of tumor cells.

腫瘍細胞のVasculogenic活性を評価するためにマトリゲルベースの​​チューブ形成アッセイ

Over the past several decades, a tube formation assay using growth factor-reduced Matrigel has been typically employed to demonstrate the angiogenic ...

Protease- and Acid-catalyzed Labeling Workflows Employing 18O-enriched Water

Stable isotopes are essential tools in biological mass spectrometry. Historically, 18O-stable isotopes have been extensively used to study the catalytic mechanisms of proteolytic enzymes1-3. With the advent of mass spectrometry-based proteomics, the enzymatically-catalyzed incorporation of 18O-atoms from stable isotopically enriched water has become a popular method to quantitatively compare protein expression levels (reviewed by Fenselau and Yao4, Miyagi and Rao5 and Ye et al.6). 18O-labeling constitutes a simple and low-cost alternative to chemical (e.g. iTRAQ, ICAT) and metabolic (e.g. SILAC) labeling techniques7. Depending on the protease utilized, 18O-labeling can result in the incorporation of up to two 18O-atoms in the C-terminal carboxyl group of the cleavage product3. The labeling reaction can be subdivided into two independent processes, the peptide bond cleavage and the carboxyl oxygen exchange reaction8. In our PALeO (protease-assisted labeling employing 18O-enriched water) adaptation of enzymatic 18O-labeling, we utilized 50% 18O-enriched water to yield distinctive isotope signatures. In combination with high-resolution matrix-assisted laser desorption ionization time-of-flight tandem mass spectrometry (MALDI-TOF/TOF MS/MS), the characteristic isotope envelopes can be used to identify cleavage products with a high level of specificity. We previously have used the PALeO-methodology to detect and characterize endogenous proteases9 and monitor proteolytic reactions10-11. Since PALeO encodes the very essence of the proteolytic cleavage reaction, the experimental setup is simple and biochemical enrichment steps of cleavage products can be circumvented. The PALeO-method can easily be extended to (i) time course experiments that monitor the dynamics of proteolytic cleavage reactions and (ii) the analysis of proteolysis in complex biological samples that represent physiological conditions. PALeO-TimeCourse experiments help identifying rate-limiting processing steps and reaction intermediates in complex proteolytic pathway reactions. Furthermore, the PALeO-reaction allows us to identify proteolytic enzymes such as the serine protease trypsin that is capable to rebind its cleavage products and catalyze the incorporation of a second 18O-atom. Such "double-labeling" enzymes can be used for postdigestion 18O-labeling, in which peptides are exclusively labeled by the carboxyl oxygen exchange reaction. Our third strategy extends labeling employing 18O-enriched water beyond enzymes and uses acidic pH conditions to introduce 18O-stable isotope signatures into peptides.

Protease- and Acid-catalyzed Labeling Workflows Employing 18O-enriched Water

Stable isotope labeling workflows employing 18O-enriched water (LeO-workflows) are versatile tools for quantitative and qualitative proteomics studies. In protease-assisted (PALeO) workflows, 18O-atoms are introduced by proteolytic cleavage and carboxyl oxygen exchange reactions mediated by proteases. In the acid-catalyzed (ALeO) workflow, 18O-atoms are introduced by carboxyl oxygen exchange at low pH.

採用プロテアーゼ及び酸触媒ラベリングワークフロー<sup&gt; 18</sup&gt; O-濃縮水

Stable isotopes are essential tools in biological mass spectrometry. Historically, 18O-stable isotopes have been extensively used to study the catalytic ...

Mouse Islet of Langerhans Isolation using a Combination of Purified Collagenase and Neutral Protease

The interrogation of beta cell gene expression and function in vitro has squarely shifted over the years from the study of rodent tumorigenic cell lines to the study of isolated rodent islets. Primary islets offer the distinct advantage that they more faithfully reflect the biology of intracellular signaling pathways and secretory responses. Whereas the method of islet isolation using tissue dissociating enzyme (TDE) preparations has been well established in many laboratories1-4, variations in the consistency of islet yield and quality from any given rodent strain limit the extent and feasibility of primary islet studies. These variations often occur as a result of the crude partially purified TDEs used in the islet isolation procedure; TDEs frequently exhibit lot-to-lot variations in activity and often require adjustments to the dose of enzyme used. A small number of reports have used purified TDEs for rodent cell isolations5, 6, but the practice is not widespread despite the routine use and advantages of purified TDEs for human islet isolations. In collaboration with VitaCyte, LLC (Indianapolis, IN), we developed a modified mouse islet isolation protocol based on that described by Gotoh7, 8, in which the TDEs are perfused directly into the pancreatic duct of mice, followed by crude tissue fractionation through a Histopaque gradient9, and isolation of purified islets. A significant difference in our protocol is the use of purified collagenase (CIzyme MA) and neutral protease (CIzyme BP) combination. The collagenase was characterized by the use of a6 fluorescence collagen degrading activity (CDA) assay that utilized fluorescently labeled soluble calf skin fibrils as substrate6. This substrate is more predictive of the kinetics of collagen degradation in the tissue matrix because it relies on native collagen as the substrate. The protease was characterized with a sensitive fluorescent kinetic assay10. Utilizing these improved assays along with more traditional biochemical analysis enable the TDE to be manufactured more consistently, leading to improved performance consistency between lots. The protocol described in here was optimized for maximal islet yield and optimal islet morphology using C57BL/6 mice. During the development of this protocol, several combinations of collagenase and neutral proteases were evaluated at different concentrations, and the final ratio of collagenase:neutral protease of 35:10 represents enzyme performance comparable to Sigma Type XI. Because significant variability in average islet yields from different strains of rats and mice have been reported, additional modifications of the TDE composition should be made to improve the yield and quality of islets recovered from different species and strains.

A detailed description of mouse islet isolation is described using the technique of in situ pancreatic ductal cannulation and perfusion of a combination of purified collagenase and neutral protease.

コラゲナーゼと中性プロテアーゼの精製の組合せを用いて単離ランゲルハンスマウスの膵島

The interrogation of beta cell gene expression and function in vitro has squarely shifted over the years from the study of rodent tumorigenic cell lines ...

Determining Membrane Protein Topology Using Fluorescence Protease Protection (FPP)

The correct topology and orientation of integral membrane proteins are essential for their proper function, yet such information has not been established for many membrane proteins. A simple technique called fluorescence protease protection (FPP) is presented, which permits the determination of membrane protein topology in living cells. This technique has numerous advantages over other methods for determining protein topology, in that it does not require the availability of multiple antibodies against various domains of the membrane protein, does not require large amounts of protein, and can be performed on living cells. The FPP method employs the spatially confined actions of proteases on the degradation of green fluorescent protein (GFP) tagged membrane proteins to determine their membrane topology and orientation. This simple approach is applicable to a wide variety of cell types, and can be used to determine membrane protein orientation in various subcellular organelles such as the mitochondria, Golgi, endoplasmic reticulum and components of the endosomal/recycling system. Membrane proteins, tagged on either the N-termini or C-termini with a GFP fusion, are expressed in a cell of interest, which is subject to selective permeabilization using the detergent digitonin. Digitonin has the ability to permeabilize the plasma membrane, while leaving intracellular organelles intact. GFP moieties exposed to the cytosol can be selectively degraded through the application of protease, whereas GFP moieties present in the lumen of organelles are protected from the protease and remain intact. The FPP assay is straightforward, and results can be obtained rapidly.

Determining Membrane Protein Topology Using Fluorescence Protease Protection FPP

Here, we present a protocol to determine the orientation and topology of integral membrane proteins in living cells. This simple protocol relies on selective protease sensitivity of chimeras between the protein of interest and GFP.

蛍光プロテアーゼ保護を使用して膜タンパク質のトポロジの決定（FPP）

The correct topology and orientation of integral membrane proteins are essential for their proper function, yet such information has not been established ...

Comet Assay as an Indirect Measure of Systemic Oxidative Stress

Higher eukaryotic organisms cannot live without oxygen; yet, paradoxically, oxygen can be harmful to them. The oxygen molecule is chemically relatively inert because it has two unpaired electrons located in different pi * anti-bonding orbitals. These two electrons have parallel spins, meaning they rotate in the same direction about their own axes. This is why the oxygen molecule is not very reactive. Activation of oxygen may occur by two different mechanisms; either through reduction via one electron at a time (monovalent reduction), or through the absorption of sufficient energy to reverse the spin of one of the unpaired electrons. This results in the production of reactive oxidative species (ROS). There are a number of ways in which the human body eliminates ROS in its physiological state. If ROS production exceeds the repair capacity, oxidative stress results and damages different molecules. There are many different methods by which oxidative stress can be measured. This manuscript focuses on one of the methods named cell gel electrophoresis, also known as &#8220;comet assay&#8221; which allows measurement of DNA breaks. If all factors known to cause DNA damage, other than oxidative stress are kept constant, the amount of DNA damage measured by comet assay is a good parameter of oxidative stress. The principle is simple and relies upon the fact that DNA molecules are negatively charged. An intact DNA molecule has such a large size that it does not migrate during electrophoresis. DNA breaks, however, if present result in smaller fragments which move in the electrical field towards the anode. Smaller fragments migrate faster. As the fragments have different sizes the final result of the electrophoresis is not a distinct line but rather a continuum with the shape of a comet. The system allows a quantification of the resulting &#8220;comet&#8221; and thus of the DNA breaks in the cell.

Comet assay measures DNA breaks, induced by different factors. If all factors (except oxidative stress) causing DNA damage are kept constant, the amount of DNA damage is a good indirect parameter of oxidative stress. The goal of this protocol is to use comet assay for indirect measurement of oxidative stress.

全身酸化ストレスの間接的な指標として、コメットアッセイ

Higher eukaryotic organisms cannot live without oxygen; yet, paradoxically, oxygen can be harmful to them. The oxygen molecule is chemically relatively ...

Identification of Kinase-substrate Pairs Using High Throughput Screening

We have developed a screening platform to identify dedicated human protein kinases for phosphorylated substrates which can be used to elucidate novel signal transduction pathways. Our approach features the use of a library of purified GST-tagged human protein kinases and a recombinant protein substrate of interest. We have used this technology to identify MAP/microtubule affinity-regulating kinase 2 (MARK2) as the kinase for a glucose-regulated site on CREB-Regulated Transcriptional Coactivator 2 (CRTC2), a protein required for beta cell proliferation, as well as the Axl family of tyrosine kinases as regulators of cell metastasis by phosphorylation of the adaptor protein ELMO. We describe this technology and discuss how it can help to establish a comprehensive map of how cells respond to environmental stimuli.

Protein phosphorylation is a central feature of how cells interpret and respond to information in their extracellular milieu. Here, we present a high throughput screening protocol using kinases purified from mammalian cells to rapidly identify kinases that phosphorylate a substrate(s) of interest.

ハイスループットスクリーニングを使用したキナーゼ基質ペアの同定


We have developed a screening platform to identify dedicated human protein kinases for phosphorylated substrates which can be used to elucidate novel ...

In Vitro Assay to Measure Phosphatidylethanolamine Methyltransferase Activity

Phosphatidylethanolamine methyltransferases are biosynthetic enzymes that catalyze the transfer of one or more methyl group(s) from S-adenosyl-L-methionine onto phosphatidylethanolamine, monomethyl-phosphatidylethanolamine, or dimethyl-phosphatidylethanolamine to give either monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine or phosphatidylcholine. These enzymes are ubiquitous in animal cells, fungi, and are also found in approximately 10% of bacteria. They fulfill various important functions in cell physiology beyond their direct role in lipid metabolism such as in insulin resistance, diabetes, atherosclerosis, cell growth, or virulence. The present manuscript reports on a simple cell-free enzymatic assay that measures the transfer of tritiated methyl group(s) from S-[Methyl-3H]adenosyl-L-methionine onto phosphatidylethanolamine using whole cell extracts as an enzyme source. The resulting methylated forms of phosphatidylethanolamine are hydrophobic and thus, can be separated from water soluble S-[Methyl-3H]adenosyl-L-methionine by organic extraction. This assay can potentially be applied to any other cell types and used to test inhibitors/drugs specific to a phosphatidylethanolamine methyltransferase of interest without the need to purify the enzyme.

In Vitro Assay to Measure Phosphatidylethanolamine Methyltransferase Activity

The present report describes an in vitro enzymatic assay to measure phosphatidylethanolamine methyltransferase activity using Leishmania cell extracts. This assay is based on the transfer of a radioactive methyl group from S-[Methyl-3H]adenosyl-L-methionine onto endogenous phosphatidylethanolamine.