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:脾脏衍生自小鼠（6-10周龄），所有动物实验均根据彼得MacCallum肿瘤中心（E486）的动物伦理准则进行。 1.准备样品。 <ol><li>准备激活小鼠NK细胞。 <ol><li> / - - （GZMB基因为null）小鼠通过阴性选择使用市售的试剂盒，从C57BL / 6或B6.GrzmB的脾脏的单细胞悬液分离主幼稚NK细胞。 </li><li>按照制造商的协议。简要地说，磁标签"非NK"细胞如T和B细胞，树突状细胞，粒细胞，巨噬细胞，并使用生物素缀合的抗体针对其特定的表面标记的红血细胞。使用磁分离与抗生物素珠粒耗尽"非NK"细胞。 </li><li>培养在24孔板中分离NK细胞5-7天以含IL-2（1000单位/毫升）的介质以700000个细胞/ ml的在37 &#160; ℃。 </li><li>可替代地，使用活化的T细胞19，初级抗原限制性CTL从OT1 T细胞受体转基因小鼠20，CTL在混合的淋巴细胞培养物21，或从上清液活化NK细胞产生的，以确定分泌GZMB 19的活性。 </li></ol></li><li>维护人类NK细胞和初级NK细胞的隔离。 <ol><li>维持人NK细胞白血病（YT细胞）在补充有10％胎牛血清罗斯威尔公园纪念研究所（RPMI）培养基。可替代地，净化从健康受试者的外周血主NK细胞和刺激2-3天在培养基含IL-2，在100单位/毫升，如前所述22。 </li></ol></li><li>代细胞裂解物。 <ol><li>在PBS（pH7.4）中洗涤细胞三次，然后悬浮于的Nonidet P-40的裂解缓冲液（0.1％NP-40，250 mM氯化钠，25毫摩尔的Hepes，2.5mM的EDTA）中。理想的是，溶解至少5×10 6个</s向上&gt; NK细胞，或1×10 7个T细胞在100μl缓冲液中。不添加蛋白酶抑制剂，因为许多丝氨酸蛋白酶的不可逆抑制剂，特别tryptases如GzmA。在冰上孵育20分钟，然后沉淀核在微量离心以15000×g离心10分钟。弃去核沉淀，上清液转移到新的管中。 </li></ol></li><li>确定的裂解物的蛋白质浓度。 <ol><li>使用市售的选择，例如，布拉德福德或二喹啉酸（BCA）的协议，并确定蛋白浓度。确保该浓度是〜2毫克/毫升最小。商店裂解物在-20℃下直到需要（粒酶的活性是稳定许多个月，在-20℃）。 </li></ol></li></ol> 2.颗粒酶B活性分析<ol><li>试剂的制备<ol><li>在DMSO准备基板（BOC-AAD-S-BZL或AC-IEPD-PNA）的10毫米股票（〜5毫克/毫升），并存储在分装在-20＆＃176℃。准备工作稀释新鲜和丢弃的每一天后，中银-AAD-S-BZL部分水解的存储DMSO。 </li><li>准备的比色试剂（5,5&#39;-二硫 - 双（2-硝基苯甲酸），DTNB）在DMSO中的250mM的股票（0.09微克/毫升），并存储在-20℃。每天准备一个新的工作稀释。该试剂只需要用于与SBzl指示符组，不pNA的衬底。 </li><li>弥补了新鲜的缓冲液（0.1M HEPES，0.05M的氯化镁2，pH值7.3），每2-3周。 </li></ol></li><li>带来DTNB和合成底物至室温。稀基板（1:16 如 1.6 ml缓冲液100微升）和DTNB（1:250， 如 10微升于2.5毫升缓冲）拌匀。 </li><li>稀至少在缓冲各样品的100微克蛋白质裂解物，以160微升的最终体积。使用多通道移液器以一式三份加入50微升每孔（〜33.3微克/孔）在一个""U""底部乙烯基96孔板。有一个"缓冲 - 只有"控制（也一式三份）。 </li><li>加入100微升稀释DTNB到样品/缓冲控制AC-IEPD-PNA工作时（省略这一步。 注:AC-IEPD-PNA由GZMB的裂解直接释放荧光PNA（ 图4B）。 </li><li>使用多道移液器快速添加50微升稀释基材每组一式三份，先从缓冲区那么任何负面的控制，预计阳性标本整理。 </li><li>将板在板读数器。测量在酶标仪在OD 405nm处为Boc - 丙氨酸 - 丙氨酸 - 天冬氨酸 - S-BZL / AC-IEPD-pNA的水解天冬氨酸酶的活性。使用动力学分析方案，以25的读数，每隔15秒（约6分钟的总阅读时间）。 </li><li>使用在每个时间点所产生的吸光率读数手动计算底物裂解的速率，如果板读取器的软件不能生成的Vmax值。 </li></ol>大3.分析TA 注意:产生被表示为最大速度的数据，定义为OD的变化随时间（MOD /分钟）。 <ol><li>分析数据，确定衬底解理，它是从吸光度变化率计算出的最大速度。自动使用平板阅读器软件，它通常有这个选项做到这一点。 </li><li>观看吸光度变化，这是在酶标仪动力学实验过程中产生的动能地块，手动后或者选择数据点，给出最陡峭的直线。对于大多数试验的最大速率是该测定的前2分钟内实现。 </li><li>减去初始OD读数从最高的OD读数在此直线和除以时间间隔。传输原始数据到Microsoft Excel /图板进行统计分析和图形显示。 </li></ol>

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The authors have nothing to disclose.

在历史上，粒酶被鉴定为细胞毒性淋巴细胞（CTL和NK细胞）能够诱导快速的凋亡性死亡的靶细胞的关键效应分子。这主要是由于GZMB的动作，这裂解靶底物分子在天门冬氨酸（D）残基，因而能由这两种裂解亲胱天蛋白酶激活的胱天蛋白酶级联，以及若干的其下游目标。然而，现在认识到，GZMB表达并不局限于淋巴细胞毒性细胞，其功能可被扩展远远超出靶细胞识别和细胞死亡。 ...

粒酶是一类在自然杀伤（NK）细胞和细胞毒性T淋巴细胞（CTL）的1的分泌溶酶体发现丝氨酸蛋白酶。在人类中（A，B，H，K和M）存在五种不同的颗粒酶和10小鼠（A - G，K，M和N）2,3。颗粒酶A和颗粒酶B（GzmA，GZMB）是最丰富的，并已被广泛研究，在人类和啮齿类动物的设置。 GZMB经典功能是细胞凋亡的诱导在与孔形成蛋白穿孔，其允许粒酶访问目标细胞胞质溶胶4一起执行靶细胞。虽然GZMB表达在细胞毒性淋巴细胞明确发现，最近的研究已经解决了各种其他GZMB表达的细胞类型，包括但不限于角质细胞5，嗜碱性粒细胞6，肥大细胞7，浆细胞样树突细胞8，和B细胞9， 10。在这种情况下，非凋亡GZMB功能进行了揭示，从参与炎症过程，组织重塑和其他免疫调节性质11-14。 鉴于一个更广泛的生物作用已被提议用于GZMB比先前怀疑，研究人员需要针对其检测可靠和专用工具。的优点是GZMB的具体要求来切割上的天冬氨酸残基，一个属性之间的真核丝氨酸蛋白酶15独特的羧基侧。小鼠，人和大鼠GZMB在结构上非常相似，但是小鼠GZMB的扩展的底物特异性不同巧妙地从人类和鼠16，这意味着某些通用基板用Asp在终端（P1）可以通过GZMB有效切割的所有三个品种，而其他底材具有更复杂的序列上游P1可能会给广泛不同的结果。在过去和最近的立terature，这一事实造成了相当大的混乱和一些实验结果的生物学意义的曲解，即使严格控制的，动力学研究试图纠正这种情况17。 在本文中，我们已经设法示出了这些点使用两个市售底物，即的Boc - 丙氨酸 - 丙氨酸 - 天冬氨酸 - SBzl和N-乙酰基异亮氨酸 - 谷氨酸 - 亲天冬氨酸 - 对硝基苯胺。的两种试剂做产生不同的反应基团以下裂解（游离巯与荧光游离对硝基酰苯胺），但这种不具有任何对蛋白水解裂解的影响。所描述的协议是一个非常古老的协议，18现代适应，但应该帮助调查人员适当地使用不同的GZMB基板，同时也提供了一个方法框架，用于检测其他颗粒酶，如GzmA和GzmH的活动。

<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.

将Boc-AAD-S- BZL基板特异于GZMB 丝氨酸蛋白酶GZMB是细胞毒性淋巴细胞（CTL和NK细胞）的主要成分，是主要负责诱导快速凋亡性死亡的靶细胞，如病毒感染或转化的细胞。这主要是由于它的底物优先用于某些特定的天冬氨酸残基中选择的蛋白质后裂解，属性与胱天，这也裂解天冬氨酸残基后，但是从一个不同的类蛋白酶，半胱氨酸蛋白酶家族15共享。在蛋白水?...

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.

比色测定的具体措施颗粒酶B蛋白水解活动:水解的Boc-丙氨酸 - 丙氨酸-ASP-S-BZL的

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 次观看.  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.

视频: 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.

荧光肽酶谱法检测蛋白酶活性

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

科研

癌症研究

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.

测量CD95死亡诱导信号传导复合物的组成和该复合物中Procaspase-8的处理

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

生物化学

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.

定性和定量测定为检测和蛋白质抗菌药物的表征

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

免疫与感染

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.

使用荧光测定法或流式细胞术测量半胱天冬酶活性

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

使用生物物理测定表征 PROTAC 中三元复合物的形成

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.

作者聚焦：评估用于表征 PROTACS 三元复合物的生物物理检测 

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).

用于评估蛋白水解靶向嵌合体（PROTACS）诱导的三元复合物形成的生物物理测定方法的发展和应用

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.

人类颗粒酶在哺乳动物细胞中高产量和经济高效表达系统

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.

用于蛋白质加工的化学发光 Western Blot 检测

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

Biological Techniques

Blot Techniques

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.

比色测定的具体措施颗粒酶B蛋白水解活动:水解的Boc-丙氨酸 - 丙氨酸-ASP-S-BZL的

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荧光肽酶谱法检测蛋白酶活性

测量CD95死亡诱导信号传导复合物的组成和该复合物中Procaspase-8的处理

定性和定量测定为检测和蛋白质抗菌药物的表征

使用荧光测定法或流式细胞术测量半胱天冬酶活性

用于评估蛋白水解靶向嵌合体（PROTACS）诱导的三元复合物形成的生物物理测定方法的发展和应用

用于评估蛋白水解靶向嵌合体（PROTACS）诱导的三元复合物形成的生物物理测定方法的发展和应用

用于评估蛋白水解靶向嵌合体（PROTACS）诱导的三元复合物形成的生物物理测定方法的发展和应用

人类颗粒酶在哺乳动物细胞中高产量和经济高效表达系统

用于蛋白质加工的化学发光 Western Blot 检测

一种用于筛选蛋白酶蛋白水解活性的荧光肽切割测定法