编制说明孵化缓冲区，缓冲区分析，并为0.6 N三氯乙酸溶液提供现成的解决方案。 FITC -酪蛋白衬底 - 这是建议分装成较小容量的到来，以避免反复冻融循环后，FITC -酪蛋白基板。如果FITC -酪蛋白衬底受到反复冻融略有增加，在后台会发生，从而降低灵敏度。等份应储存在-20 ° C，避光。每个样品，空白或控制的反应需要20μLFITC -酪蛋白衬底。冻结/解冻，以及大力搅拌或摇晃，可能会导致的FITC标记的分离在一个较高的背景阅读并导致更少的基板可用于蛋白酶切割产生的酪蛋白。 异硫氰酸荧光素（FITC）控制解决方案的FITC标记的控制，可检测缓冲液中重组，以适当的浓度。这个解决方案应该是新鲜和避光。 最后的胰蛋白酶控制解决方案 - 1毫米的盐酸中加入100μL胰蛋白酶，蛋白酶控制（产品编号：T 6567）的小瓶。混合简要保证胰蛋白酶溶解。新增900孵化缓冲液拌匀。另外，其他的缓冲区可以使用，如果需要检测。最后工作的胰蛋白酶的浓度为20微克/μL。当准备编写胰蛋白酶控制解决方案，结合了正确的孵育缓冲液（1 9部分缓冲区的部分酸化胰蛋白酶）的酸性胰蛋白酶溶液的等分。在酸性条件下储存的胰蛋白酶，增加胰蛋白酶的稳定性。胰蛋白酶控制的解决方案是对温度敏感的，而不是在低浓度的稳定。 请审查工具包中的这些准备的解决方案的存储和稳定的技术公告。 程序<ol><li>这是最简单的控制，空白和样品都在同一时间准备。对于每一个测试样本中，孵育缓冲液20μL，20μLFITC -酪蛋白基板，和10μL的测试样品添加微量离心管。蛋白酶活性高的测试样品，样品稀释液可能是必需的。 </li><li>加入20μl孵育缓冲液，20μLFITC -酪蛋白基板，控制样品和10μL的离心管，准备适当的控制样本（见控制样本）。 </li><li>离心管加入20μL，孵育缓冲液，20μLFITC -酪蛋白基板，和10μL超纯水制备空白样品。 </li><li>轻轻地混合在37 ° C黑暗环境中为60分钟，每管和孵化。小心太大力不要混用，因为过多的动荡可能会导致高的荧光背景，并减少了检测的灵敏度。 注：孵育时间可能会延长至24小时，以提高灵敏度。要小心，不要超过24小时，FITC -酪蛋白可能开始降低，导致高荧光背景。 </li><li>孵育后，添加150μL的0.6 N氯乙酸的解决方案，以每个离心管。 TCA是具有很强的腐蚀性，应穿着适当的防护设备，而处理。 </li><li>轻轻混匀，于37孵育° C 30分钟的黑暗环境​​中。 </li><li> 10分钟，离心10,000 × G.管上清含有溶于酸，FITC标记的片段，并使用荧光测量。 </li></ol>荧光测量这些方法可以缩小或根据现有的仪器的要求。比较适当的控制样本编制的标准曲线，减去空白样品，从每个测试样本值（FLUtest）（FLUblank）的荧光读数。 比色皿<ol><li>吸取10μL上清（步骤7）和1毫升到一个合适的比色皿测定缓冲区，并轻轻混匀。注：上清，检测缓冲的解决方案可能会储存在2-8℃，在黑暗中长达24小时前测量的荧光。 </li><li>记录485 nm处的荧光强度与激发和发射波长为535 nm监测。 </li></ol>多孔板<ol><li>吸取10μL上清（步骤7）和1毫升的检测到一个合适的试管或小瓶缓冲区，轻轻混匀注：上清，检测缓冲的解决方案，在2-8 ° C，最多可存储在黑暗中24小时前测量的荧光。 </li><li>将200μL以及黑色96孔板。记录485 nm处的荧光强度与激发和发射波长为535 nm监测。 或者</li></ol><ol><li>吸取2μL上清液（步骤7）和B的含量的200μLuffer成黑色96孔板以及。注：上清，检测缓冲的解决方案可能会储存在2-8℃，在黑暗中长达24小时前测量的荧光。 </li><li>记录485 nm处的荧光强度与激发和发射波长为535 nm监测。 </li></ol>控制样本胰蛋白酶控制解决方案可用于确认检测是正确执行，以确定检测限，或创建一个通用的标准曲线。对于不同的检测，特异性蛋白酶，它是建议准备控制在适当的孵化缓冲液中含有特异性蛋白酶。 检测的检测限是蛋白酶，产生显着高于空白样品中获得的价值的荧光读。取决于仪器的灵敏度，检测限会有所不同。胰蛋白酶控制解决方案的系列稀释，可用于生成控制解决方案。 阅读等于120％，与空白样品中获得的价值被认为是重要的。常规检测胰蛋白酶的5纳克的限制与此过程中获得。建议每次检测至少有一个5吴胰蛋白酶控制运行。胰蛋白酶的浓度为0.5微克/毫升与控制解决方案所需的5胰蛋白酶吴检测结果。一个40倍稀释液，胰蛋白酶控制解决方案（20微克/毫升）的结果在0.5微克/毫升的控制解决方案，即胰蛋白酶39部分孵化缓冲控制解决方案的一部分。 <img src="/files/ftp_upload/1514/figure1.jpg" alt="图1" /> 图1（胰蛋白酶的活性标准曲线）：胰蛋白酶控制解决方案（20微克/毫升），也可用于生成通过连续稀释的标准曲线（见图1） 。每个控制样品的荧光读数减去空白样品荧光读值从每个控制样品（FLUcontrol）（FLUblank）已得到纠正。 典型的胰蛋白酶“（产品代码T 6567）的标准曲线，使用该试剂盒和控制范围从0.15微克/毫升（1.5纳克）至2.5微克/毫升（25毫微克）的样品。这条曲线是由以下所描述的过程，孵化时间30分钟步骤4产生。在多孔板，荧光测量。 异硫氰酸荧光素作为一种可能的校准仪器的控制（见相应的制造商的指示）或FITC标记信号的线性范围的确定。

<ol>
	<li>Twining, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescein+Isothiocyanate-Labeled+Casein+Assay+for+Proteolytic+Enzymes.">Fluorescein Isothiocyanate-Labeled Casein Assay for Proteolytic Enzymes.</a> Anal. Biochem. 143, 30-34 (1984).</li></ol>

作者是产生本文中使用的试剂和工具的Sigma Aldrich公司的雇员。

我们刚刚例如检测使用蛋白酶荧光检测试剂盒来检测生物样品中蛋白酶的活性。该试剂盒进行了优化，检测发现在生理应用的蛋白酶的多元化。它是适用于检测丝氨酸，半胱氨酸的金属，和天门冬氨酸蛋白酶;然而，可能需要修改，​​以检测某些特定的蛋白酶。

<table border="1">
<tr>
<td>Material Name</td>
<td>Type</td>
<td>Company</td>
<td>Catalogue Number</td>
<td>Comments (optional)</td>
</tr>
<tr>
<td><a href="http://www.sigmaaldrich.com/catalog/ProductDetail.do?lang=en&N4=PF0100|SIGMA&N5=SEARCH_CONCAT_PNO|BRAND_KEY&F=SPEC&cm_sp=affiliate-_-Jove-_-12346">Protease Fluorescent Detection Kit</a></td>
<td>&nbsp;</td>
<td>Sigma Aldrich</td>
<td>PF0100</td>
<td>&nbsp;</td>
<tr>
<td><a href="http://www.sigmaaldrich.com/catalog/ProductDetail.do?lang=en&N4=H1758|SIGMA&N5=SEARCH_CONCAT_PNO|BRAND_KEY&F=SPEC&cm_sp=affiliate-_-Jove-_-12347">Hydrochloric Acid</a></td>
<td>&nbsp;</td>
<td>Sigma Aldrich</td>
<td>H1758</td>
<td>&nbsp;</td>
</table>

use of the protease fluorescent detection kit to determine protease activity

蛋白酶荧光检测试剂盒提供了现成使用的试剂，检测蛋白酶活性的存在。这个简单的实验，检测蛋白酶活性使用酪蛋白为底物异硫氰酸荧光素（FITC）标记。 成更小的碎片，没有在酸性条件下沉淀的FITC标记的酪蛋白底物裂解蛋白酶活性的结果。蛋白酶样品和基板的潜伏期后，与另外三氯乙酸（TCA）的酸化反应。然后将混合物离心未消化形成一种颗粒较小，酸溶液中剩余的可溶性片段的衬底。上清瓦解和FITC标记的片段荧光测量。 所描述的套件程序检测胰蛋白酶蛋白酶浓度约为0.5微克/毫升（5胰蛋白酶吴检测）的控制。这种敏感性可以增加一个潜伏期较长时间，最长为24小时。在离心管进行检测和荧光检测使用的是试管或多孔板提供程序。

Watch this Scientific Journal Video about Use of the Protease Fluorescent Detection Kit to Determine Protease Activity at JoVE.com

Use of the Protease Fluorescent Detection Kit to Determine Protease Activity

蛋白酶荧光检测试剂盒是专为使用蛋白酶活性荧光测量。它也是适合的蛋白酶污染的痕量检测。该方法是基于对专利配方的一个FITC标记的酪蛋白底物的水解hydroysis。

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 ...

use-protease-fluorescent-detection-kit-to-determine-protease

Nanyang Technological University

Research

JoVE Journal

Biology

28.1K 次观看.  Sigma Aldrich. 蛋白酶荧光检测试剂盒是专为使用蛋白酶活性荧光测量。它也是适合的蛋白酶污染的痕量检测。该方法是基于对专利配方的一个FITC标记的酪蛋白底物的水解hydroysis。

视频: Use of the Protease Fluorescent Detection Kit to Determine Protease Activity

Purifying Plasmid DNA from Bacterial Colonies Using the Qiagen Miniprep Kit

Plasmid DNA purification from E. coli is a core technique for molecular cloning. Small scale purification (miniprep) from less than 5 ml of bacterial culture is a quick way for clone verification or DNA isolation, followed by further enzymatic reactions (polymerase chain reaction and restriction enzyme digestion). Here, we video-recorded the general procedures of miniprep through the QIAGEN's QIAprep 8 Miniprep Kit, aiming to introducing this highly efficient technique to the general beginners for molecular biology techniques. The whole procedure is based on alkaline lysis of E. coli cells followed by adsorption of DNA onto silica in the presence of high salt. It consists of three steps: 1) preparation and clearing of a bacterial lysate, 2) adsorption of DNA onto the QIAprep membrane, 3) washing and elution of plasmid DNA. All steps are performed without the use of phenol, chloroform, CsCl, ethidium bromide, and without alcohol precipitation. It usually takes less than 2 hours to finish the entire procedure.

使用Qiagen公司的小量制备试剂盒的细菌菌落纯化质粒DNA

Plasmid DNA purification from E. coli is a core technique for molecular cloning. Small scale purification (miniprep) from less than 5 ml of bacterial ...

科研

生物学

Immunohistochemistry: Paraffin Sections Using the Vectastain ABC Kit from Vector Labs

Immunohistochemistry (IHC) is a valuable technique utilized to localize/visualize protein expression in a mounted tissue section using specific antibodies. There are two methods: the direct and indirect method. In this experiment, we will only describe the use of indirect IHC staining. Indirect IHC staining utilizes highly specific primary and biotin-conjugated secondary antibodies. Primary antibodies are utilized to discretely identify proteins of interest by binding to a specific epitope, while secondary antibodies subtract for non-specific background staining and amplify signal by forming complexes to the primary antibody. Slides can either be generated from frozen sections, or paraffin embedded sections mounted on glass slides. In this protocol, we discuss the preparation of paraffin-embedded sections by dewaxing, hydration using an alcohol gradient, heat induced antigen retrieval, and blocking of endogenous peroxidase activity and non-specific binding sites. Some sections are then stained with antibodies specific for T cell marker CD8 and while others are stained for tyrosine hydroxylase. The slides are subsequently treated with appropriate secondary antibodies conjugated to biotin, then developed utilizing avidin-conjugated horseradish peroxidase (HRP) with Diaminiobenzidine (DAB) as substrate. Following development, the slides are counterstained for contrast, and mounted under coverslips with permount. After adequate drying, these slides are then ready for imaging.

免疫组化：石蜡切片从Vector实验室使用Vectastain ABC试剂盒

Immunohistochemistry (IHC) is a valuable technique utilized to localize/visualize protein expression in a mounted tissue section using specific ...

Labeling Stem Cells with Fluorescent Dyes for non-invasive Detection with Optical Imaging

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo labeling of stem cells with a fluorescent dye, subsequent intravenous injection of the labeled cells and visualization of their accumulation in specific target organs or pathologies. The presented technique demonstrates how we label human mesenchymal stem cells (hMSC) by simple incubation with the lipophilic fluorescent dye DiD (C67H103CIN2O3S) and how we label human embryonic stem cells (hESC) with the FDA approved fluorescent dye Indocyanine Green (ICG). The uptake mechanism is via adherence and diffusion of the lypophilic dye across the phospholipid cell membrane bilayer. The labeling efficiency is usually improved if the cells are incubated with the dye in serum-free media as opposed to incubation in serum-containing media. Furthermore, the addition of the transfection agent Protamine Sulfate significantly improves contrast agent uptake.

This video shows techniques for labeling of human embryonic stem cells and mesenchymal stem cells with fluorescent dyes. This technique can be used for an in vivo tracking of transplanted stem cells with optical imaging and for histopathological correlations with fluorescence microscopy.

与非侵入性检测荧光染料标记的干细胞与光学成像

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo ...

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

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 activity assay may be used as a standardized procedure to determine the activity of proteases, which is what we do during our quality control procedures. In this assay, casein acts as a substrate. When the protease we are testing digests casein, the amino acid tyrosine is liberated along with other amino acids and peptide fragments. Folin and Ciocalteus Phenol, or Folin's reagent primarily reacts with free tyrosine to produce a blue colored chromophore, which is quantifiable and measured as an absorbance value on the spectrophotometer. The more tyrosine that is released from casein, the more the chromophores are generated and the stronger the activity of the protease. Absorbance values generated by the activity of the protease are compared to a standard curve, which is generated by reacting known quantities of tyrosine with the F-C reagent to correlate changes in absorbance with the amount of tyrosine in micromoles. From the standard curve the activity of protease samples can be determined in terms of Units, which is the amount in micromoles of tyrosine equivalents released from casein per minute.
To view this article in Chinese, <a href="https://jove-com.remotexs.ntu.edu.sg/index/details.stp?ID=910">click here</a>

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 activity assay may be used as a standardized procedure to determine the activity of proteases.

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 ...

Detection of Protein Ubiquitination

Ubiquitination, the covalent attachment of the polypeptide ubiquitin to target proteins, is a key posttranslational modification carried out by a set of three enzymes. They include ubiquitin-activating enzyme E1, ubiquitin-conjugating enzyme E2, and ubiquitin ligase E3. Unlike to E1 and E2, E3 ubiquitin ligases display substrate specificity. On the other hand, numerous deubiquitylating enzymes have roles in processing polyubiquitinated proteins. Ubiquitination can result in change of protein stability, cellular localization, and biological activity. Mutations of genes involved in the ubiquitination/deubiquitination pathway or altered ubiquitin system function are associated with many different human diseases such as various types of cancer, neurodegeneration, and metabolic disorders. The detection of altered or normal ubiquitination of target proteins may provide a better understanding on the pathogenesis of these diseases. &nbsp;Here, we describe protocols to detect protein ubiquitination in cultured cells in vivo and test tubes in vitro. These protocols are also useful to detect other ubiquitin-like small molecule modification such as sumolyation and neddylation.

Ubiquitination is a key posttranslational modification carried out by a set of three enzymes. Mutations of genes involved in this modification are associated with many different human diseases. Here, we describe protocols to detect protein ubiquitination in cultured cells in vivo and test tubes in vitro.

蛋白泛素化的检测

Ubiquitination, the covalent attachment of the polypeptide ubiquitin to target proteins, is a key posttranslational modification carried out by a set of ...

Direct Detection of the Acetate-forming Activity  of the Enzyme Acetate Kinase

Acetate kinase, a member of the acetate and sugar kinase-Hsp70-actin (ASKHA) enzyme superfamily1-5, is responsible for the reversible phosphorylation of acetate to acetyl phosphate utilizing ATP as a substrate. Acetate kinases are ubiquitous in the Bacteria, found in one genus of Archaea, and are also present in microbes of the Eukarya6. The most well characterized acetate kinase is that from the methane-producing archaeon Methanosarcina thermophila7-14. An acetate kinase which can only utilize PPi but not ATP in the acetyl phosphate-forming direction has been isolated from Entamoeba histolytica, the causative agent of amoebic dysentery, and has thus far only been found in this genus15,16.
In the direction of acetyl phosphate formation, acetate kinase activity is typically measured using the hydroxamate assay, first described by Lipmann17-20, a coupled assay in which conversion of ATP to ADP is coupled to oxidation of NADH to NAD+ by the enzymes pyruvate kinase and lactate dehydrogenase21,22, or an assay measuring release of inorganic phosphate after reaction of the acetyl phosphate product with hydroxylamine23. Activity in the opposite, acetate-forming direction is measured by coupling ATP formation from ADP to the reduction of NADP+ to NADPH by the enzymes hexokinase and glucose 6-phosphate dehydrogenase24.
Here we describe a method for the detection of acetate kinase activity in the direction of acetate formation that does not require coupling enzymes, but is instead based on direct determination of acetyl phosphate consumption. After the enzymatic reaction, remaining acetyl phosphate is converted to a ferric hydroxamate complex that can be measured spectrophotometrically, as for the hydroxamate assay. Thus, unlike the standard coupled assay for this direction that is dependent on the production of ATP from ADP, this direct assay can be used for acetate kinases that produce ATP or PPi.

A method for the determination of acetate kinase activity is described. This assay utilizes a direct reaction for determining enzyme activity and kinetics of acetate kinase in the acetate-forming direction with different phosphoryl acceptors. Furthermore, this method can be utilized for assaying other acetyl phosphate or acetyl-CoA utilizing enzymes.

醋酸形成醋酸酶激酶活性的直接检测

Acetate kinase, a member of the acetate and sugar kinase-Hsp70-actin (ASKHA) enzyme superfamily1-5, is responsible for the reversible phosphorylation of ...

Mouse Sperm Cryopreservation and Recovery using the I&middot;Cryo Kit

Thousands of new genetically modified (GM) strains of mice have been created since the advent of transgenesis and knockout technologies. Many of these valuable animals exist only as live animals, with no backup plan in case of emergency. Cryopreservation of embryos can provide this backup, but is costly, can be a lengthy procedure, and generally requires a large number of animals for success. Since the discovery that mouse sperm can be successfully cryopreserved with a basic cryoprotective agent (CPA) consisting of 18&#37; raffinose and 3&#37; skim milk, sperm cryopreservation has become an acceptable and cost-effective procedure for archiving, distributing and recovery of these valuable strains.
 Here we demonstrate a newly developed I&bull;Cryo kit for mouse sperm cryopreservation. Sperm from five commonly-used strains of inbred mice were frozen using this kit and then recovered. Higher protection ratios of sperm motility (&gt; 60&#37;) and rapid progressive motility (&gt; 45&#37;) compared to the control (basic CPA) were seen for sperm frozen with this kit in 5 inbred mouse strains. Two cell stage embryo development after IVF with the recovered sperm was improved consistently in all 5 mouse strains examined. Over a 1.5 year period, 49 GM mouse lines were archived by sperm cryopreservation with the I&bull;Cryo kit and later recovered by IVF.

Here we demonstrate the newly developed I&#x2022;Cryo kit for mouse sperm cryopreservation. Two-cell stage embryo development with frozen-thawed sperm was improved consistently in 5 mouse strains with the use of this kit. Over a 1.5 year period, 49 genetically modified mouse lines were archived by sperm cryopreservation with the I&#x2022;Cryo kit and later successfully recovered by IVF.

精子冷冻保存和恢复使用I ·冷冻包

Thousands of new genetically modified (GM) strains of mice have been created since the advent of transgenesis and knockout technologies.  Many of these ...

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 ...