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 &#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

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="https://jove-com.remotexs.ntu.edu.sg/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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76.1K 次观看.  Sigma Aldrich.  ChineseVersion 

视频: 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.

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.

使用Sigma的提取物N -安培组织PCR试剂盒小鼠组织的快速基因分型

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 ...

科研

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.

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 ...

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.

用于测量的活性测定大肠埃希氏菌诱导赖氨酸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.

基于一个基质胶管形成实验，以评估对肿瘤细胞的血管生成活性

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

Assaying the Kinase Activity of LRRK2 in vitro

Leucine Rich Repeat Kinase 2 (LRRK2) is a 2527 amino acid member of the ROCO family of proteins, possessing a complex, multidomain structure including a GTPase domain (termed ROC, for Ras of Complex proteins) and a kinase domain1. The discovery in 2004 of mutations in LRRK2 that cause Parkinson's disease (PD) resulted in LRRK2 being the focus of a huge volume of research into its normal function and how the protein goes awry in the disease state2,3. Initial investigations into the function of LRRK2 focused on its enzymatic activities4-6. Although a clear picture has yet to emerge of a consistent alteration in these due to mutations, data from a number of groups has highlighted the importance of the kinase activity of LRRK2 in cell death linked to mutations7,8. Recent publications have reported inhibitors targeting the kinase activity of LRRK2, providing a key experimental tool9-11. In light of these data, it is likely that the enzymatic properties of LRRK2 afford us an important window into the biology of this protein, although whether they are potential drug targets for Parkinson's is open to debate.
A number of different approaches have been used to assay the kinase activity of LRRK2. Initially, assays were carried out using epitope tagged protein overexpressed in mammalian cell lines and immunoprecipitated, with the assays carried out using this protein immobilised on agarose beads4,5,7. Subsequently, purified recombinant fragments of LRRK2 in solution have also been used, for example a GST tagged fragment purified from insect cells containing residues 970 to 2527 of LRRK212. Recently, Dani&euml;ls et al. reported the isolation of full length LRRK2 in solution from human embryonic kidney cells, however this protein is not widely available13. In contrast, the GST fusion truncated form of LRRK2 is commercially available (from Invitrogen, see table 1 for details), and provides a convenient tool for demonstrating an assay for LRRK2 kinase activity. Several different outputs for LRRK2 kinase activity have been reported. Autophosphorylation of LRRK2 itself, phosphorylation of Myelin Basic Protein (MBP) as a generic kinase substrate and phosphorylation of an artificial substrate - dubbed LRRKtide, based upon phosphorylation of threonine 558 in Moesin - have all been used, as have a series of putative physiological substrates including &alpha;-synuclein, Moesin and 4-EBP14-17. The status of these proteins as substrates for LRRK2 remains unclear, and as such the protocol described below will focus on using MBP as a generic substrate, noting the utility of this system to assay LRRK2 kinase activity directed against a range of potential substrates.

Assaying the Kinase Activity of LRRK2 in vitro

Leucine Rich Repeat Kinase 2 is a large multidomain kinase, mutations in which are the most common genetic cause of Parkinson's disease. Analysis of the kinase activity of this protein has proven to be a crucial tool in understanding the biology and dysfunction of this protein. In this paper, in vitro assaying of the kinase activity of LRRK2 and a selection of its mutants is described, providing an experimental system to examine phosphorylation of putative substrates and potential dysfunction of LRRK2 in disease.

含量测定的LRRK2的激酶活性在体外</em

Leucine Rich Repeat Kinase 2 (LRRK2) is a 2527 amino acid member of the ROCO family of proteins, possessing a complex, multidomain structure including a ...

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.

蛋白酶和酸催化的贴标工作流程，用人<sup&gt; 18</sup&gt; O-富氧水

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.

小鼠胰岛的朗格汉斯的分离纯化的胶原酶和中性蛋白酶结合使用

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.

确定膜蛋白拓扑使用荧光蛋白酶保护（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.

彗星试验系统性氧化应激的间接测量

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.

激酶底物对鉴定使用高通量筛选


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

Sigma的非特异性蛋白酶活性测定 - 酪蛋白作为基质

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