在开始检测之前，我们需要确保正确地准备以下试剂： <ol><li> 50毫米的磷酸钾缓冲液，pH值7.5。准备用11.4毫克/毫升，钾phospate二元，纯净水，用1M盐酸调节pH值的三水。这个解决方案是在37 ° C，使用前放置。 </li><li>一个0.65％重量/体积的酪蛋白溶液，混合，在50 mM磷酸钾缓冲6.5毫克/毫升酪蛋白准备。逐渐升高溶液温度80-85℃，约10分钟轻轻搅拌，直到均匀的分散实现。这是非常重要的是不要熬的解决方案。必要时用NaOH和HCl调整pH值。 </li><li>一个110毫米的三氯乙酸溶液，用纯净水稀释一个6.1N股票1:55准备。三氯乙酸是一种强酸，应小心处理。 </li><li> 0.5毫米福林Ciolcaltea的，或福林酚试剂，这是解决方案，将与酪氨酸反应，生成一个可衡量的的颜色变化，将直接关系到蛋白酶的活性。福林酚试剂是一种酸，应谨慎处理。 </li><li> 500毫米碳酸钠的解决方案，准备使用纯净水53毫克/毫升的anyhydrous碳酸钠。 一种酶稀释剂的解决方案，其中包括10 mM磷酸钠，醋酸钙5MM醋酸缓冲液，pH值7.5，在37 ° C。这个解决方案是我们用来溶解固体蛋白酶样品或稀释酶解决方案。 </li><li> 1.1毫米L -酪氨酸的标准储备溶液。准备使用0.2 mg / ml的L -酪氨酸，在纯净水和加热，轻轻酪氨酸溶解。与酪蛋白，这种解决方案不开锅。 L -酪氨酸的标准冷却到室温。该解决方案将进一步被摊薄，使我们的标准曲线。 </li><li>蛋白酶的解决方案。使用前，溶解蛋白酶的酶稀释液在第6步准备的解决方案。 </li></ol>如果有必要，使用了预定的活动奠定坚实的蛋白酶样品，这是用酶稀释液0.1-0.2单位/ ml溶解。该解决方案作为一个质量控制检测的阳性对照和验证的计算，我们将执行确定酶的活性。 设立蛋白酶含​​量及标准曲线<ol><li>要开始这个实验中，找到合适的，将举行约15毫升的小瓶。对于每个将被测试的酶，需要4瓶。一小瓶将被用来作为一个空白，和其他三人将被用来检测三个稀释的蛋白酶活性。三稀释检查时，对对方的最终计算是有用的。 <ol><li>每一套四瓶，添加0.65％酪蛋白溶液5mls。让他们在37℃水浴平衡5分钟左右彗星。 </li><li>添加不同的酶液，将被测试的测试样品瓶三个卷，但不是空白。混合纷飞和37 ° C孵育整整10分钟。蛋白酶活性和酪氨酸相应的解放，在这个孵化时间是将被测量和比较试验样品。 </li></ol></li><li>这10分钟的潜伏期后，每管加入5毫升的TCA试剂终止反应。然后，添加适当的酶液量，以每管，甚至空白，使酶液，每管最终成交量是1毫升。这样做是考虑到酶本身的吸光度值，以确保在每管的最后体积等于。的解决方案在37 ° C下30分钟。 在这30分钟的孵育，您可能需要设置您的酪氨酸标准稀释。使用6 DRAM小瓶（DRAM小瓶，可取代聚丙烯管），可以轻松容纳8 MLS。到六瓶，加入MLS以下几卷的1.1毫米酪氨酸的标准储备溶液：0.05，0.10，0.20，0.40，0.50。不添加任何酪氨酸标准的空白。不纯的测试样本，将产生的颜色变化不大，可能需要降低标准。一旦酪氨酸标准溶液已被添加，添加适当体积的纯净水标准，把2毫升的量。 </li><li>经过30分钟的孵化，筛选每个测试解决方案，并使用0.45微米的聚醚砜注射器过滤器的空白。过滤器是必需的，从样本中删除任何不溶物。 <ol><li>过滤测试样品2毫升和空白滤液4 DRAM小瓶，可容纳至少8 MLS。可以使用同一类型的小瓶，其中的标准准备。 </li><li>所有的小瓶含有的标准和标准的空白，添加碳酸钠5mls。为了达到最佳效果，随即添加1毫升福林的试剂。 </li><li>添加碳酸钠规范福林试剂除了创建任何pH值下降。 </li><li>添加碳酸钠，测试样品和工商业污水附加费t空白。这些解决方案成为后的碳酸钠除了阴天。新增的福林试剂，将主要免费酪氨酸反应。 </li><li>混合纷飞的DRAM小瓶，并在37 ° C孵育30分钟。 </li></ol>此潜伏期后，你应该注意到，标准的色阶与酪氨酸添加量相关的;酪氨酸的最高浓度出现的最黑暗的。您也可以通知我们的测试样本中明显的颜色变化。这些解决方案2mls到合适的试管中使用0.45微米的聚醚砜注射器过滤器过滤。现在进行检测，你可以继续分光光度计记录我们的吸光度值。 </li></ol>测吸光度，计算酶的活性<ol><li>一个使用660nm波长的分光光度计测量样品的吸光度。光路设置为1cm。记录的标准，标准的空白，不同的测试样本，和测试空白的吸光度值。一旦所有的数据都已经收集，可以创建标准曲线。为了生成的曲线，在吸光度之间的​​标准和标准的空白差异，必须计算。这是吸光度值占酪氨酸量标准的解决方案。这个简单的计算后，建立标准曲线，使用图形程序绘制Y轴的改变，在我们的标准的吸光度，对我们的5 X轴的标准量在微摩尔。 <table style="width: 416px; height: 118px;" border="0"><tbody><tr><th style="text-align:center">酪氨酸标准卷</th><th style="text-align:center"> uMoles酪氨酸</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>已输入的数据点后，生成一个最适合和相应的缓坡方程的行。 查找计算测试样品的吸光度和测试空白的吸光度之间的​​差异的测试样品的光吸收变化。插入缓坡方程的一个测试样品的吸光度值和解决，将导致酪氨酸解放微摩尔在这个特殊的蛋白水解反应。为了得到每单位/毫升的活性酶，执行下列计算： （发布umole酪氨酸等效）×（11） 单位/毫升酶= _________________________________________ （1）X（10）×（2） 11 =总体积（毫升）的测定 10 =时间法（分钟）为单位定义 1 =酶的酶量（毫升）使用 2 =体积（毫升）比色法测定 以微摩尔酪氨酸从缓坡方程获得等值发布的数量和乘以MLS的检测，这在我们的例子是11mls总量。的酶量的检测中使用，分成三个其他数量这个值：的检测，其中我们10分钟跑的时候，是不同的（的使用1毫升），的比色法检测毫升的量，这可能你的比色皿不同的基础上。我们用2毫升。 </li><li>按时间分为酪氨酸微摩尔分钟产量蛋白酶活性的所谓“单位”的测量。我们可以取消了单位体积中的分子和分母的测量，留在单位/ ml酶的活性measurment。为了确定了坚实的蛋白酶的酶稀释液稀释后的样品的活性，分裂我们的活动，在单位/毫升浓度毫克/毫升。原本在这个实验中使用的固体，留给我们单位/ mg活动。 单位/毫升酶 单位/ mg固体= ____________________ 固体毫克/毫升酶 </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>

我们刚刚向您展示了如何使用Sigma的普遍蛋白酶活性测定分析蛋白酶活性。此外，这种检测，以确保我们蛋白酶精确确定活动之前，您将收到您的实验，他们是有用的。正如您所看到的，在执行此过程时，记得要热的酪蛋白和酪氨酸的解决方案，慢慢地熬他们最重​​要的。此外，它的关键，以备您的标准和每个测试样本，你有不同的空白。

<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 &amp;amp; Ciocalteu&#039;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

蛋白酶打破肽键。在实验室中，它往往是必要的，来衡量和/或比较蛋白酶的活性。 Sigma的非特异性的蛋白酶活性检测可作为一个标准化的程序，以确定蛋白酶的活性，这是我们在我们的质量控制程序。在这个实验中，酪蛋白作为基板。当我们正在测试的蛋白酶消化酪蛋白，氨基酸酪氨酸是沿解放与其他的氨基酸和肽片段。福林和Ciocalteus苯酚，或福林的试剂主要是反应与自由的酪氨酸产生蓝色生色，这是量化和分光光度计的吸光度值来衡量。从酪蛋白释放更多的酪氨酸，更生色团产生较强的蛋白酶的活性。蛋白酶的活动所产生的吸光度值相比，这是已知数量的酪氨酸反应与FC试剂关联吸光度的变化量在微摩尔酪氨酸产生的标准曲线。从标准曲线的活性蛋白酶样品，可以在单位，这是在微摩尔每分钟从酪蛋白释放的酪氨酸等值金额方面确定。 要查看这篇文章在中国， <a href="http://jove.com/index/details.stp?ID=910">请点击这里</a>

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

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

蛋白酶打破肽键。在实验室中，它往往是必要的，来衡量和/或比较蛋白酶的活性。 Sigma的非特异性的蛋白酶活性检测可作为一个标准化的程序，以确定蛋白酶的活性。

Sigma的非特异性蛋白酶活性测定 - 酪蛋白作为基质

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

The Determination of Protease Specificity in Mouse Tissue Extracts by MALDI-TOF Mass Spectrometry: Manipulating PH to Cause Specificity Changes

Proteases have several biological functions, including protein activation/inactivation and food digestion. Identifying protease specificity is important for revealing protease function. The method proposed in this study determines protease specificity by measuring the molecular weight of unique substrates using Matrix-assisted Laser Desorption/Ionization Time-of-Flight (MALDI-TOF) mass spectrometry. The substrates contain iminobiotin, while the cleaved site consists of amino acids, and the spacer consists of polyethylene glycol. The cleaved substrate will generate a unique molecular weight using a cleaved amino acid. One of the merits of this method is that it may be carried out in one pot using crude samples, and it is also suitable for assessing multiple samples. In this article, we describe a simple experimental method optimized with samples extracted from mouse lung tissue, including tissue extraction, placement of digestive substrates into samples, purification of digestive substrates under different pH conditions, and measurement of the substrates&#39; molecular weight using MALDI-TOF mass spectrometry. In summary, this technique allows for the identification of protease specificity in crude samples derived from tissue extracts using MALDI-TOF mass spectrometry, which may easily be scaled up for multiple sample processing.

Here, we present a protocol to determine protease specificity in crude mouse tissue extracts using MALDI-TOF spectrometry.

MALDI 质谱法测定小鼠组织提取物蛋白酶特异性: 操作 PH 值导致特异性改变

Proteases have several biological functions, including protein activation/inactivation and food digestion. Identifying protease specificity is important ...

科研

生物化学

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 ...

癌症研究

A Fluorogenic Peptide Cleavage Assay to Screen for Proteolytic Activity: Applications for coronavirus spike protein activation

Enveloped viruses such as coronaviruses or influenza virus require proteolytic cleavage of their fusion protein to be able to infect the host cell. Often viruses exhibit cell and tissue tropism and are adapted to specific cell or tissue proteases. Moreover, these viruses can introduce mutations or insertions into their genome during replication that may affect the cleavage, and thus can contribute to adaptations to a new host. Here, we present a fluorogenic peptide cleavage assay that allows a rapid screening of peptides mimicking the cleavage site of viral fusion proteins. The technique is very flexible and can be used to investigate the proteolytic activity of a single protease on many different substrates, and in addition, it also allows exploration of the activity of multiple proteases on one or more peptide substrates. In this study, we used peptides mimicking the cleavage site motifs of the coronavirus spike protein. We tested human and camel derived Middle East Respiratory Syndrome coronaviruses (MERS-CoV) to demonstrate that single and double substitutions in the cleavage site can alter the activity of furin and dramatically change cleavage efficiency. We also used this method in combination with bioinformatics to test furin cleavage activity of feline coronavirus spike proteins from different serotypes and strains. This peptide-based method is less labor- and time intensive than conventional methods used for the analysis of proteolytic activity for viruses, and results can be obtained within a single day.

We present a fluorogenic peptide cleavage assay that allows a rapid screening of the proteolytic activity of proteases on peptides representing the cleavage site of viral fusion peptides. This method can also be used on any other amino acid motif within a protein sequence to test for the protease activity.

一种用于蛋白质溶解活性的荧光肽裂解检测: 冠状病毒穗蛋白活化的应用

Enveloped viruses such as coronaviruses or influenza virus require proteolytic cleavage of their fusion protein to be able to infect the host cell. Often ...

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.

蛋白酶荧光检测试剂盒，用它来确定蛋白酶活性

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

生物学

The Importance of Correct Protein Concentration for Kinetics and Affinity Determination in Structure-function Analysis 

In this study, we explore the interaction between the bovine cysteine protease inhibitor cystatin B and a catalytically inactive form of papain (Fig. 1), a plant cysteine protease, by real-time label-free analysis using Biacore X100. Several cystatin B variants with point mutations in areas of interaction with papain, are produced. For each cystatin B variant we determine its specific binding concentration using calibration-free concentration analysis (CFCA) and compare the values obtained with total protein concentration as determined by A280. After that, the kinetics of each cystatin B variant binding to papain is measured using single-cycle kinetics (SCK). We show that one of the four cystatin B variants we examine is only partially active for binding. This partial activity, revealed by CFCA, translates to a significant difference in the association rate constant (ka) and affinity (KD), compared to the values calculated using total protein concentration. Using CFCA in combination with kinetic analysis in a structure-function study contributes to obtaining reliable results, and helps to make the right interpretation of the interaction mechanism.

The Importance of Correct Protein Concentration for Kinetics and Affinity Determination in Structure-function Analysis

We apply label-free protein interaction analysis using Biacore X100 for structure-function analysis of the binding of several cystatin B mutants to papain through kinetic characterization. Calibration-free concentration analysis (CFCA) measures the concentration of protein with retained binding activity without the need for a standard curve. We show that confirmation of concentrations using CFCA increases the reliability of the kinetic analysis and that kinetic constants can reliably be determined even if the activity of a recombinant protein is reduced.

正确的蛋白浓度的动力学结构与功能分析和亲和力测定的重要性

In this study, we explore the interaction between the bovine cysteine protease inhibitor cystatin B and a catalytically inactive form of papain (Fig. 1), ...

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

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.

基于刀豆球蛋白A的沉降测定法，用于测量葡聚糖磷酸酶的底物结合

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

Detection of Functional Matrix Metalloproteinases by Zymography

Matrix metalloproteinases (MMPs) are zinc-containing endopeptidases. They degrade proteins by cleavage of peptide bonds. More than twenty MMPs have been identified and are separated into six groups based on their structure and substrate specificity (collagenases, gelatinases, membrane type [MT-MMP], stromelysins, matrilysins, and others). MMPs play a critical role in cell invasion, cartilage degradation, tissue remodeling, wound healing, and embryogenesis. They therefore participate in both normal processes and in the pathogenesis of many diseases, such as rheumatoid arthritis, cancer, or chronic obstructive pulmonary disease1-6. Here, we will focus on MMP-2 (gelatinase A, type IV collagenase), a widely expressed MMP. We will demonstrate how to detect MMP-2 in cell culture supernatants by zymography, a commonly used, simple, and yet very sensitive technique first described in 1980 by C. Heussen and E.B. Dowdle7-10. This technique is semi-quantitative, it can therefore be used to determine MMP levels in test samples when known concentrations of recombinant MMP are loaded on the same gel11.
Solutions containing MMPs (e.g. cell culture supernatants, urine, or serum) are loaded onto a polyacrylamide gel containing sodium dodecyl sulfate (SDS; to linearize the proteins) and gelatin (substrate for MMP-2). The sample buffer is designed to increase sample viscosity (to facilitate gel loading), provide a tracking dye (bromophenol blue; to monitor sample migration), provide denaturing molecules (to linearize proteins), and control the pH of the sample. Proteins are then allowed to migrate under an electric current in a running buffer designed to provide a constant migration rate. The distance of migration is inversely correlated with the molecular weight of the protein (small proteins move faster through the gel than large proteins do and therefore migrate further down the gel). After migration, the gel is placed in a renaturing buffer to allow proteins to regain their tertiary structure, necessary for enzymatic activity. The gel is then placed in a developing buffer designed to allow the protease to digest its substrate. The developing buffer also contains p-aminophenylmercuric acetate (APMA) to activate the non-proteolytic pro-MMPs into active MMPs. The next step consists of staining the substrate (gelatin in our example). After washing the excess dye off the gel, areas of protease digestion appear as clear bands. The clearer the band, the more concentrated the protease it contains. Band staining intensity can then be determined by densitometry, using a software such as ImageJ, allowing for sample comparison.

This protocol describes an activity-based assay for detecting matrix metalloproteinases in culture supernatants or body fluids.

酶谱法检测功能的基质金属蛋白酶

Matrix metalloproteinases (MMPs) are zinc-containing endopeptidases. They degrade proteins by cleavage of peptide bonds. More than twenty MMPs have been ...

Direct Detection of Isolevuglandins in Tissues Using a D11 scFv-Alkaline Phosphatase Fusion Protein and Immunofluorescence

Isolevuglandins (IsoLGs) are highly reactive gamma ketoaldehydes formed from H2-isoprostanes through lipid peroxidation and crosslink proteins leading to inflammation and various diseases including hypertension. Detection of IsoLG accumulation in tissues is crucial in shedding light on their involvement in the disease processes. However, measurement of IsoLGs in tissues is extremely difficult, and currently available tools, including mass spectrometry analysis, are laborious and extremely expensive. Here we describe a novel method for in situ detection of IsoLGs in tissues using alkaline phosphatase-conjugated D11 ScFv and a recombinant phage-display antibody produced in E. coli by immunofluorescent microscopy. Four controls were used for validating the staining: (1) staining with and without D11, (2) staining with bacterial periplasmic extract with the alkaline phosphatase linker, (3) irrelevant scFV antibody staining, and (4) competitive control with IsoLG prior to the staining. We demonstrate the effectiveness of the alkaline phosphatase-conjugated D11 in both human and mouse tissues with or without hypertension. This method will likely serve as an important tool to study the role of IsoLGs in a wide variety of disease processes.

This article provides a detailed methodology for the measurement of isolevuglandins in tissues by immunofluorescence using alkaline phosphatase-conjugated ScFv D11 antibody. Hypertension models in both mice and humans are used to explain the step-by-step procedures and fundamental principles associated with isolevuglandin measurement in tissue samples.

使用 D11 scFv-碱性磷酸酶融合蛋白和免疫荧光直接检测组织中的异左旋蛋白

Isolevuglandins (IsoLGs) are highly reactive gamma ketoaldehydes formed from H2-isoprostanes through lipid peroxidation and crosslink proteins leading to ...

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

Source: Jaimes, J. A., et al. <a href="https://app.jove.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.

Sigma的非特异性蛋白酶活性测定 - 酪蛋白作为基质

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