This work received support through grant HA 6136/1-1 from the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) to MH. JAT is supported by Program and Project Grants from the National Health and Medical Research Council of Australia.

注：脾臓をマウス（6〜10週齢）に由来し、すべての動物実験は、ピーター·マッカラム癌センター（E486）の動物倫理ガイドラインに従って行った。 サンプルの調製。 <ol><li>活性化したマウスNK細胞の調製。 <ol><li>市販のキットを用いたネガティブ選択により（GZMB遺伝子ヌル）マウス- / - C57BL / 6またはB6.GrzmBの脾臓の単一細胞懸濁液からの一次ナイーブなNK細胞を単離する。 </li><li>メーカーのプロトコルに従ってください。簡単に説明すると、磁気的TおよびB細胞、樹状細胞、顆粒球、マクロファージ、およびそれらの特定の表面マーカーに対するビオチン標識抗体を用いて赤血球などの「非NK」細胞を標識する。 「非NK」細胞を枯渇させる抗ビオチンビーズと磁気分離を使用してください。 </li><li>培養物を24ウェルプレートに70万細胞/ mlのIL-2（1,000 U / ml）を含む培地中で5~7日間、NK細胞を単離する37時&#160; °C。 </li><li>あるいは、使用は19 T細胞、OT1 T細胞受容体トランスジェニックマウス20からの一次抗原制限CTLは、CTL分泌GZMB 19の活性を決定するために、活性化NK細胞からの混合リンパ球培養21、または上清中に生成された活性化した。 </li></ol></li><li>ヒトNK細胞株および一次NK細胞の単離の維持。 <ol><li> 10％ウシ胎児血清を補充したロズウェルパーク記念研究所（RPMI）培地中のヒトNK白血病（YT細胞）を維持する。代替的に、健康な被験者の末梢血から一次NK細胞を精製し、以前に22を説明したように100 U / mlのIL-2を含む培地中で2~3日間刺激する。 </li></ol></li><li>細胞溶解物を生成する。 <ol><li> PBS（pH7.4）中で細胞を3回洗浄し、次いで、ノニデットP-40溶解緩衝液に懸濁（0.1％NP-40、250のNaCl、25mMのヘペス、2.5mMのEDTA）。理想的には、5×10 6の最小値を溶解</sNK細胞、または100μlの緩衝液中の1×10 7 T細胞&gt;アップ。多くはセリンプロテアーゼの不可逆的阻害剤であるように、プロテアーゼ阻害剤を追加して、そのようなGZMAなどの特定のトリプターゼでしないでください。 20分間氷上でインキュベートし、次いで10分間15000×gで微量で核をペレット化。核ペレットを廃棄し、新しいチューブに上清を移す。 </li></ol></li><li>溶解物のタンパク質濃度を決定します。 <ol><li>そのようなブラッドフォードやビシンコニン酸（BCA）、などの選択肢の市販のプロトコルを使用して、タンパク質濃度を決定する。濃度が〜2 mg / mlの最小であることを確認してください。必要になるまで-20℃で保存溶解物（グランザイム活動は-20℃で何ヶ月も安定している）。 </li></ol></li></ol> 2.グランザイムB活性アッセイ<ol><li>試薬の調製<ol><li> -20＆＃でアリコートに10 mMのDMSO（〜5 mg / mlの）における基板の株式（のBoc-AAD-S-BzlのまたはAC-IEPD-PNA）とストアを準備する176; C。たての作業希釈液を調製及びBoc-AAD-S-Bzlのは、部分的に、DMSO中のストレージに加水分解するとして、それぞれの日の後に捨てる。 </li><li> -20℃で250 mMのDMSO中の比色試薬（5,5&#39;-ジチオ - ビス（2-ニトロ安息香酸）、DTNB）の株式（0.09グラム/ ml）およびストアを準備します。毎日新鮮な作業希釈を準備します。この試薬は、のみSBzlインジケータグループ、しないのpNAを有する基板に必要です。 </li><li>新鮮な緩衝液（0.1M HEPES、0.05 MのMgCl 2、pH7.3）で2〜3週間毎を構成している。 </li></ol></li><li>室温にDTNBと合成基質を持参してください。基質（1：16、 例えばバッファの1.6ミリリットルで100μl）を希釈し、DTNB（1：250、 例えば 2.5ミリリットルバッファ内の10μl）を、よく混ぜる。 </li><li> 160μlの最終容量までバッファ内の各サンプルの100μgのタンパク質溶解物の最低希釈する。マルチチャンネルピペットを使用すると、 &#39;&#39; U &#39;&#39;底のビニール96ウェルプレートに（〜33.3 /ウェル）三重にウェルあたり50μlを添加する。（また、三重で）「バッファ専用」コントロールを含めます。 </li><li>のAc-IEPD-PNAを扱う際のサンプル/バッファ制御に希釈しDTNB100μlのを追加します（この手順を省略します。 注：GZMBにより交流-IEPD-pNAの切断は、直接蛍光性PNA（ 図4B）をリリースします。 </li><li>その後緩衝液を用いて任意の負の制御を開始し、期待陽性サンプルを終え、すぐに三重の各セットに希釈した基質50μlのを追加するためにマルチチャンネルピペットを使用してください。 </li><li>プレートリーダーでプレートを置きます。 OD 405 nmでマイクロプレートリーダーでのBoc-ALA-ALA-Aspを-S-Bzlの/ AC-IEPD-pNAの加水分解によるのAspアーゼ活性を測定する。 25の測定値、15秒毎（約6分の合計読み取り時間）を取る、運動アッセイプロトコルを使用してください。 </li><li>プレートリーダーのソフトウェアがVmaxの値を生成できない場合、手動で基質切断の速度を計算するために、各時点で生成された吸光度測定値を使用する。 </li></ol>ダ3.分析TA 注：生成されたデータは、時間（MOD /分）を超えるODの変化として定義された最大速度として提示される。 <ol><li>データを分析するために、吸光度の変化率から計算される基質切断の最大速度を決定する。通常は、このオプションを持って、プレートリーダーソフトウェアを使用して、これを自動的に行う。 </li><li>代わりに手動で、プレートリーダー上で動的アッセイ中に生成される吸光度の変化の速度論的プロットを、表示した後に最も急な直線を与えるデータポイントを選択します。ほとんどのアッセイの最大レートは、アッセイの最初の2分以内に達成される。 </li><li>時間間隔によって、この直線と除算で最高OD読み取りから初期ODの読みを引く。統計分析およびグラフィック表示のためのMicrosoft Excel /グラフパッドに生データを転送します。 </li></ol>

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The authors have nothing to disclose.

歴史的にグランザイムは、標的細胞の急速なアポトーシス死を誘導することができる細胞傷害性リンパ球（CTLおよびNK細胞）の重要なエフェクター分子として同定された。これは主に、アスパラギン酸（D）残基で標的基質分子を切断し、したがって両方の切断プロカスパーゼによってカスパーゼカスケードを活性化することができるだけでなく、それらの下流の標的のいくつかあったGZMBの作...

グランザイムは、ナチュラルキラー（NK）細胞および細胞傷害性Tリンパ球（CTL）1の分泌リソソームに見出されるセリンプロテアーゼのファミリーである。五つの異なるグランザイムは、ヒト（A、B、H、KとM）で存在し、そしてマウスにおける10（A - G、K、M及びN）2,3。グランザイムAおよびグランザイムB（GZMA、GZMB）が最も豊富であり、広範囲に人間と齧歯類の​​設定で研究されてきた。 GZMBの古典的な機能は、標的細胞の細胞質ゾル4にアクセスするためのグランザイムを可能にする孔形成タンパク質、パーフォリンと組み合わせて実行される、標的細胞におけるアポトーシスの誘導である。 GZMB発現が明確に細胞傷害性リンパ球において見出されているが、最近の研究では、好塩基球6、肥満細胞7、形質細胞様樹状細胞8、およびB細胞9を含むが、ケラチノサイト5に限定されるものではなく、他のGZMBを発現する種々の細胞型に対処されてきた、 10。この文脈では、非アポトーシスGZMB機能は炎症プロセス、組織リモデリングおよび他の免疫調節特性11-14の参加の範囲が明らかになった。 より広範な生物学的役割は、以前に疑われるよりもGZMBのために提案されていることを考えると、研究者は、その検出のための信頼性の高い、特定のツールが必要です。有利なのアスパラギン残基のカルボキシル側で切断するGZMBの特定の要件、真核生物のセリンプロテアーゼ15の中でユニークな特性である。マウス、ヒトおよびラットGZMBしかしマウスGZMBの拡張された基質特異性端子（P1）のAspを有する特定のジェネリック基質は、GZMBによって効率的に切断することができることを意味し、ヒトおよびラット16の両方とは微妙に異なるが、構造的に非常に類似しているすべての3種から、上流のP1のより複雑な配列をもつ他の基材に対し、広く多様な結果を与える可能性があります。過去と最近の李両方でterature、この事実は、注意深く制御、動態試験状況17を修正しようとしているにもかかわらず、いくつかの実験結果の生物学的意義のかなりの混乱や誤解を引き起こした。 本論文では、2つの市販の基板、すなわちのBoc-ALA-ALA-Aspを-SBzlとN-アセチルイルのGlu-Proの-ASP-p-ニトロアニリドを使用して、これらのポイントを説明しようとしてきた。 2つの試薬は、切断（蛍光無料パラニトロアニ対遊離スルフヒドリル）以下の異なる反応基を生成行いますが、これはタンパク質分解的切断には影響何もありません。記載されているプロトコルは、非常に古いプロトコル18の近代的な適応ですが、また、そのようなGZMAやGzmHなど他グランザイム、の活性を検出するための方法論的な枠組みを提供しながら、研究者は、適切に異なるGZMB基板を使用するように助けるべきである。

<table><tbody><tr><td>Boc-Ala-Ala-Asp-S-Bzl</td><td>SM Biochemicals LLC, CA</td><td>SMSB05</td><td>Granzyme B substrate (mouse and human)</td></tr><tr><td>Ac-IEPD-pNA&amp;nbsp;</td><td>SM Biochemicals LLC, CA</td><td>SMPNA009</td><td>Granzyme B substrate (only human)</td></tr><tr><td>N-&amp;#945;-CBZ-L-lysine-S-Bzl</td><td>Sigma-Aldrich</td><td>C3647</td><td>Granzyme A substrate</td></tr><tr><td>Suc-Phe-Leu-Phe-S-Bzl</td><td>SM Biochemicals LLC, CA</td><td>SB025</td><td>Granzyme H substrate</td></tr><tr><td>5,5&amp;rsquo;-dithio-bis(2-nitrobenzoic acid)&amp;nbsp;</td><td>Sigma-Aldrich</td><td>D8130</td><td>DTNB, Ellman&amp;rsquo;s Reagent</td></tr><tr><td>NK cell isolation kit II mouse</td><td>Miltenyi Biotec GmbH</td><td>130-096-892</td><td>negative selection kit</td></tr><tr><td>NK cell isolation kit human</td><td>Miltenyi Biotec GmbH</td><td>130-092-657</td><td>negative selection kit</td></tr><tr><td>Plate reader</td><td>Biorad iMark</td><td></td><td>Biorad Microplate Manager Software Version MPM6.3</td></tr><tr><td>Serocluster U-bottom vinyl 96-well plate</td><td>Corning, MA, USA</td><td>2797</td><td></td></tr></tbody></table>

a colorimetric assay that specifically measures granzyme b proteolytic activity: hydrolysis of boc-ala-ala-asp-s-bzl

The serine protease Granzyme B (GzmB) mediates target cell apoptosis when released by cytotoxic T lymphocytes (CTL) or natural killer (NK) cells. GzmB is the most studied granzyme in humans and mice and therefore, researchers need specific and reliable tools to study its function and role in pathophysiology. This especially necessitates assays that do not recognize proteases such as caspases or other granzymes that are structurally or functionally related. Here, we apply GzmB&#8217;s preference for cleavage after aspartic acid residues in a colorimetric assay using the peptide thioester Boc-Ala-Ala-Asp-S-Bzl. GzmB is the only mammalian serine protease capable of cleaving this substrate. The substrate is cleaved with similar efficiency by human, mouse and rat GzmB, a property not shared by other commercially available peptide substrates, even some that are advertised as being suitable for this purpose. This protocol is demonstrated using unfractionated lysates from activated NK cells or CTL and is also suitable for recombinant proteases generated in a variety of prokaryotic and eukaryotic systems, provided the correct controls are used. This assay is a highly specific method to ascertain the potential pro-apoptotic activity of cytotoxic molecules in mammalian lymphocytes, and of their recombinant counterparts expressed by a variety of methodologies.

のBoc-AAD-S-Bzlの基板は、GZMBに特異的である セリンプロテアーゼGZMBは細胞傷害性リンパ球（CTLおよびNK細胞）の主成分であり、そのようなウイルス感染または形質転換細胞などの標的細胞に迅速なアポトーシス死を誘導するのに主に責任がある。これはまた、アスパラギン残基の後に切断するが、プロテアーゼの異なるクラス、システインプロテアーゼファミ...

Watch this Scientific Journal Video about A Colorimetric Assay that Specifically Measures Granzyme B Proteolytic Activity: Hydrolysis of Boc-Ala-Ala-Asp-S-Bzl at JoVE.com

A Colorimetric Assay that Specifically Measures Granzyme B Proteolytic Activity: Hydrolysis of Boc-Ala-Ala-Asp-S-Bzl

We describe a simple, quantitative colorimetric assay that specifically measures the proteolytic activity of human, mouse or rat Granzyme B (GzmB). This protocol can be easily adapted for determining protease activity of other granule serine proteases by the hydrolysis of other synthetic peptide substrates with an appropriate recognition sequence.

のBoc-ALA-ALA-Aspを-S-Bzlのの加水分解：具体的にグランザイムBタンパク質分解活性を測定する比色アッセイ

The serine protease Granzyme B (GzmB) mediates target cell apoptosis when released by cytotoxic T lymphocytes (CTL) or natural killer (NK) cells. GzmB is ...

a-colorimetric-assay-that-specifically-measures-granzyme-b

Universidad de Cartagena UDEC

Research

JoVE Journal

Chemistry

13.0K ビュー.  Peter MacCallum Cancer Centre. We describe a simple, quantitative colorimetric assay that specifically measures the proteolytic activity of human, mouse or rat Granzyme B (GzmB). This protocol can be easily adapted for determining protease activity of other granule serine proteases by the hydrolysis of other synthetic peptide substrates with an appropriate recognition sequence.

ビデオ: A Colorimetric Assay that Specifically Measures Granzyme B Proteolytic Activity: Hydrolysis of Boc-Ala-Ala-Asp-S-Bzl

Detection of Protease Activity by Fluorescent Peptide Zymography

The purpose of this method is to measure the proteolytic activity of complex biological samples. The samples are separated by molecular weight using electrophoresis through a resolving gel embedded with a degradable substrate. This method differs from traditional gel zymography in that a quenched fluorogenic peptide is covalently incorporated into the resolving gel instead of full length proteins, such as gelatin or casein. Use of the fluorogenic peptides enables direct detection of proteolytic activity without additional staining steps. Enzymes within the biological samples cleave the quenched fluorogenic peptide, resulting in an increase in fluorescence. The fluorescent signal in the gels is then imaged with a standard fluorescent gel scanner and quantified using densitometry. The use of peptides as the degradable substrate greatly expands the possible proteases detectable with zymographic techniques.

Here, we present a detailed protocol for a modified zymographic technique in which fluorescent peptides are used as the degradable substrate in place of native proteins. Electrophoresis of biological samples in fluorescent peptide zymograms enables detection of a wider range of proteases than previous zymographic techniques.

蛍光ペプチド ザイモグラフィーによってプロテアーゼ活性の検出

The purpose of this method is to measure the proteolytic activity of complex biological samples. The samples are separated by molecular weight using ...

がん研究

Measuring Composition of CD95 Death-Inducing Signaling Complex and Processing of Procaspase-8 in this Complex

Extrinsic apoptosis is mediated by the activation of death receptors (DRs) such as CD95/Fas/APO-1 or tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-receptor 1/receptor 2 (TRAIL-R1/R2). Stimulation of these receptors with their cognate ligands leads to the assembly of the death-inducing signaling complex (DISC). DISC comprises DR, the adaptor protein Fas-associated protein with death domain (FADD), procaspases-8/-10, and cellular FADD-like interleukin (IL)-1&#946;-converting enzyme-inhibitory proteins (c-FLIPs). The DISC serves as a platform for procaspase-8 processing and activation. The latter occurs via its dimerization/oligomerization in the death effector domain (DED) filaments assembled at the DISC. 
Activation of procaspase-8 is followed by its processing, which occurs in several steps. In this work, an established experimental workflow is described that allows the measurement of DISC formation and the processing of procaspase-8 in this complex. The workflow is based on immunoprecipitation techniques supported by western blot analysis. This workflow allows careful monitoring of different steps of procaspase-8 recruitment to the DISC and its processing and is highly relevant for investigating molecular mechanisms of extrinsic apoptosis.

Here, an experimental workflow is presented that enables the detection of caspase-8 processing directly at the death-inducing signaling complex (DISC) and determines the composition of this complex. This methodology has broad applications, from unraveling the molecular mechanisms of cell death pathways to the dynamic modeling of apoptosis networks.

CD95死誘導シグナル複合体の組成とプロスパスパーゼ-8の処理の測定

Extrinsic apoptosis is mediated by the activation of death receptors (DRs) such as CD95/Fas/APO-1 or tumor necrosis factor-related apoptosis-inducing ...

生化学

Qualitative and Quantitative Assays for Detection and Characterization of Protein Antimicrobials

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods to screen for target enzymes prior to investing greater research effort and resources. The goal of this protocol is to demonstrate two complementary assays for conducting these initial evaluations. The microslide diffusion assay provides an initial or simple detection screen to enable the qualitative and rapid assessment of proteolytic activity against an array of both viable and heat-killed bacterial target substrates. As a counterpart, the increased sensitivity and reproducibility of the dye-release assay provides a quantitative platform for evaluating and comparing environmental influences affecting the hydrolytic activity of protein antimicrobials. The ability to label specific heat-killed cell culture substrates with Remazol brilliant blue R dye expands this capability to tailor the dye-release assay to characterize enzymatic activity of interest.

Here, we present protocols to rapidly screen and characterize enzymes for antimicrobial activity. The microslide diffusion assay and the dye-release assay utilize target bacterial substrates for qualitative and quantitative enzymatic activity evaluation.

定性的および定量的検出のためのアッセイおよびタンパク質の抗菌薬のキャラクタリゼーション

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods ...

免疫・感染学

Measuring Caspase Activity Using a Fluorometric Assay or Flow Cytometry

The activation of cysteine proteases, known as caspases, remains an important process in multiple forms of cell death. Caspases are critical initiators and executioners of apoptosis, the most studied form of programmed cell death. Apoptosis occurs during developmental processes and is a necessary event in tissue homeostasis. Pyroptosis is another form of cell death that utilizes caspases and is a critical process in activating the immune system through the activation of the inflammasome, which results in the release of members of the interleukin-1 (IL-1) family. To assess caspase activity, target substrates can be assessed. However, sensitivity can be an issue when examining single cells or low-level activity. We demonstrate how a fluorogenic substrate can be used with a population-based assay or single-cell assay by flow cytometry. With proper controls, different amino acid sequences can be used to identify which caspases are active. Using these assays, the simultaneous loss of the inhibitors of apoptosis proteins upon tumor necrosis factor (TNF) stimulation has been identified, which primarily induces apoptosis in macrophages rather than other forms of cell death.

The present protocol describes two methods to measure caspase activity through a fluorogenic substrate using flow cytometry or a spectrofluorometer.

蛍光アッセイまたはフローサイトメトリーを使用したカスパーゼ活性の測定

The activation of cysteine proteases, known as caspases, remains an important process in multiple forms of cell death. Caspases are critical initiators ...

生物物理学的アッセイを用いたPROTACにおける三元複合体形成の特性評価

The Development and Application of Biophysical Assays for Evaluating Ternary Complex Formation Induced by Proteolysis Targeting Chimeras (PROTACS)

E3 ligases and proteins targeted for degradation can be induced to form complexes by heterobifunctional molecules in a multi-step process. The kinetics and thermodynamics of the interactions involved contribute to efficiency of ubiquitination and resulting degradation of the protein. Biophysical techniques such as surface plasmon resonance (SPR), biolayer interferometry (BLI), and isothermal titration calorimetry (ITC) provide valuable information that can be used in the optimization of those interactions. Using two model systems, a biophysical assay tool kit for understanding the cooperativity of ternary complex formation and the impact of the &#39;hook effect&#39; on binding kinetics was established. In one case, a proteolysis targeting chimera (PROTAC) molecule that induced ternary complex formation between Brd4BD2 and VHL was evaluated. The heterobifunctional molecule, MZ1, has nM affinities for both the Brd4BD2 protein (SPR KD = 1 nM, ITC KD = 4 nM) and the VHL complex (SPR KD = 29 nM, ITC KD = 66 nM). For this system, robust SPR, BLI, and ITC assays were developed that reproduced published results demonstrating the cooperativity of ternary complex formation. In the other case, a molecule that induced ternary complexes between a 46.0 kDa protein, PPM1D, and cereblon [CRBN (319-442)] was studied. The heterobifunctional molecule, BRD-5110, has an SPR KD = 1 nM for PPM1D but much weaker binding against the truncated CRBN (319-442) complex (SPR KD= ~ 3 &#181;M). In that case, the binding for CRBN in SPR was not saturable, resulting in a &#34;hook-effect&#34;. Throughput and reagent requirements for SPR, BLI, and ITC were evaluated, and general recommendations for their application to PROTAC projects were provided.

著者スポットライト:PROTACS三元複合体の特性評価のための生物物理学的アッセイの評価 

Here we describe protocols for the biophysical characterization of ternary complex formation induced by proteolysis targeting chimeras (PROTACS) that involve the ubiquitin ligases Von Hippel-Lindau E3 ligase (VHL) and Cereblon (CRBN). Biophysical methods illustrated herein include surface plasmon resonance (SPR), biolayer interferometry (BLI), and isothermal titration calorimetry (ITC).

キメラを標的としたタンパク質分解による三元複合体形成評価のための生物物理学的アッセイの開発と応用(PROTACS)

E3 ligases and proteins targeted for degradation can be induced to form complexes by heterobifunctional molecules in a multi-step process. The kinetics ...

A High Yield and Cost-efficient Expression System of Human Granzymes in Mammalian Cells

When cytotoxic T lymphocytes (CTL) or natural killer (NK) cells recognize tumor cells or cells infected with intracellular pathogens, they release their cytotoxic granule content to eliminate the target cells and the intracellular pathogen. Death of the host cells and intracellular pathogens is triggered by the granule serine proteases, granzymes (Gzms), delivered into the host cell cytosol by the pore forming protein perforin (PFN) and into bacterial pathogens by the prokaryotic membrane disrupting protein granulysin (GNLY). To investigate the molecular mechanisms of target cell death mediated by the Gzms in experimental in-vitro settings, protein expression and purification systems that produce high amounts of active enzymes are necessary. Mammalian secreted protein expression systems imply the potential to produce correctly folded, fully functional protein that bears posttranslational modification, such as glycosylation. Therefore, we used a cost-efficient calcium precipitation method for transient transfection of HEK293T cells with human Gzms cloned into the expression plasmid pHLsec. Gzm purification from the culture supernatant was achieved by immobilized nickel affinity chromatography using the C-terminal polyhistidine tag provided by the vector. The insertion of an enterokinase site at the N-terminus of the protein allowed the generation of active protease that was finally purified by cation exchange chromatography. The system was tested by producing high levels of cytotoxic human Gzm A, B and M and should be capable to produce virtually every enzyme in the human body in high yields.

We describe here a cost-efficient granzyme expression system using HEK293T cells that produces high yields of pure, fully glycosylated and enzymatically active protease.

哺乳動物細胞におけるヒトグランザイムの高収量およびコスト効率の高い発現システム

When cytotoxic T lymphocytes (CTL) or natural killer (NK) cells recognize tumor cells or cells infected with intracellular pathogens, they release their ...

生物学

Chemiluminescent Western Blot Assay for Protein Processing

Source: Hillert-Richter, L. K. et al., <a href="https://appjove.unicartagenaproxy.elogim.com/v/62842">Measuring Composition of CD95 Death-Inducing Signaling Complex and Processing of Procaspase-8 in this Complex.</a> J. Vis. Exp. (2021).
This video describes the chemiluminescence-based western blotting of immunoprecipitated lysates. The technique helps determine protein processing and activation. The chemiluminescent signal detected is proportional to the level of protein processing during its activation.

タンパク質プロセッシングのための化学発光ウェスタンブロットアッセイ

Source: Hillert-Richter, L. K. et al., Measuring Composition of CD95 Death-Inducing Signaling Complex and Processing of Procaspase-8 in this Complex. J. ...

JoVE Encyclopedia of Experiments

生物学的手法

ブロット技術

A Fluorogenic Peptide Cleavage Assay to Screen the Proteolytic Activity of Proteases

Source: Jaimes, J. A., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/58892">A Fluorogenic Peptide Cleavage Assay to Screen for Proteolytic Activity: Applications for coronavirus spike protein activation.</a> J. Vis. Exp. (2019).
This video demonstrates an assay to screen for the proteolytic activity of proteases using fluorogenic peptides. The protease recognizes its cleavage site on the peptide, cleaving it and separating the quencher from the fluorophore, enabling its fluorescence emission. The fluorescence signal is detected and analyzed to check for the cleavage efficiency of different peptide variants.

のBoc-ALA-ALA-Aspを-S-Bzlのの加水分解：具体的にグランザイムBタンパク質分解活性を測定する比色アッセイ

要約

さらに動画を探す

この動画の章

蛍光ペプチドザイモグラフィーによってプロテアーゼ活性の検出

CD95死誘導シグナル複合体の組成とプロスパスパーゼ-8の処理の測定

定性的および定量的検出のためのアッセイおよびタンパク質の抗菌薬のキャラクタリゼーション

蛍光アッセイまたはフローサイトメトリーを使用したカスパーゼ活性の測定

キメラを標的としたタンパク質分解による三元複合体形成評価のための生物物理学的アッセイの開発と応用(PROTACS)

キメラを標的としたタンパク質分解による三元複合体形成評価のための生物物理学的アッセイの開発と応用(PROTACS)

キメラを標的としたタンパク質分解による三元複合体形成評価のための生物物理学的アッセイの開発と応用(PROTACS)

哺乳動物細胞におけるヒトグランザイムの高収量およびコスト効率の高い発現システム

タンパク質プロセッシングのための化学発光ウェスタンブロットアッセイ

プロテアーゼのタンパク質分解活性をスクリーニングするための蛍光性ペプチド切断アッセイ

のBoc-ALA-ALA-Aspを-S-Bzlのの加水分解：具体的にグランザイムBタンパク質分解活性を測定する比色アッセイ

要約

さらに動画を探す

この動画の章

蛍光ペプチド ザイモグラフィーによってプロテアーゼ活性の検出

CD95死誘導シグナル複合体の組成とプロスパスパーゼ-8の処理の測定

定性的および定量的検出のためのアッセイおよびタンパク質の抗菌薬のキャラクタリゼーション

蛍光アッセイまたはフローサイトメトリーを使用したカスパーゼ活性の測定

キメラを標的としたタンパク質分解による三元複合体形成評価のための生物物理学的アッセイの開発と応用(PROTACS)

キメラを標的としたタンパク質分解による三元複合体形成評価のための生物物理学的アッセイの開発と応用(PROTACS)

キメラを標的としたタンパク質分解による三元複合体形成評価のための生物物理学的アッセイの開発と応用(PROTACS)

哺乳動物細胞におけるヒトグランザイムの高収量およびコスト効率の高い発現システム

タンパク質プロセッシングのための化学発光ウェスタンブロットアッセイ

プロテアーゼのタンパク質分解活性をスクリーニングするための蛍光性ペプチド切断アッセイ

蛍光ペプチドザイモグラフィーによってプロテアーゼ活性の検出