作者感谢用友科学为生命奖霍华德·休斯医学研究所的啊

1。准备磁带<ol><li>清洁的正面和背面板使用Invitrogen公司Nupage NOVEX微小胶体录像带，或其他商业微小胶体录像带。 </li><li>准备约1-3毫升的塑料环氧树脂，并根据制造商的指示，拌匀。 </li><li>适用于环氧树脂内缘的前面板上，两个板块接触时，磁带装配。 </li><li>通过背面板的底部缝滑入了一块过滤纸或纸板，装配时的录像带，以防止多余的环氧密封开幕。取出滤纸，只要这两个板块组装。 </li><li>将沿着一个密封的边缘粘结剂剪辑，并让磁带通宵坐在环氧树脂完全固化。 </li><li>电工胶带背面板的底部密封的缝隙。 </li></ol> 2。浇注胶<ol><li>侧臂瓶125毫升，加入以下内容：我总体积为9毫升试剂级水（表意文字- H 2 O）的2.25毫升4X Tris碱分离胶缓冲液pH值8.70，30.8％，丙烯酰胺/双丙烯酰胺3.50毫升，3.25毫升类型。 </li><li>德气体烧瓶的侧臂连接到一个真空装置，并放置在一个倒置的烧瓶上方塞10分钟的解决方案。 </li><li>转移去瓦斯的解决方案，以50毫升的一次性聚丙烯离心管9毫升，并添加过硫酸铵（APS）的10％ammounium 30μL和6μL氮，氮，N“，N-四甲基乙二胺（TEMED）和混合纷飞的解决方案，以避免引入气泡。 </li><li>立即倒入磁带的解决方案，允许它运行下来的录像带一侧，以防止形成凝胶板之间的任何气泡。录像带，以填补前（短）板的顶部边缘约2厘米。 </li><li>轻轻地添加0.5毫升-1 - 丁醇饱和1X解决凝胶缓冲，让凝胶聚合​​1小时。 </li></ol> 3。浇注浓缩胶<ol><li>加入125毫升侧臂瓶：4X Tris碱浓缩胶缓冲液pH值6.80，0.40毫升30.8％丙烯酰胺/双丙烯酰胺，表意文字-H 2 O 1.85毫升3毫升的总量为0.75毫升以下。 </li><li>德气体10分钟，如在第2步中所述的解决方案。 </li><li>而脱气浓缩胶溶液，倒入1 - 丁醇与1X解析解决凝胶顶部的凝胶缓冲饱和的。一旦与表意文字-H 2 O冲洗解决凝胶顶部倒掉水，并用吸水或滤纸滤纸吸收剩余的液体。 </li><li>取出3毫升的脱气的解决方案，并添加9μl和10％APS和2μLTEMED拌匀。 </li><li>很快倒在录像带上的解决方案，允许它运行录像带一侧，直到它附近的短前凝胶板的顶部边缘。 </li><li>清洁凝胶梳插入凝胶盒的顶部，磁带上的​​粘合剂剪辑放置在梳举行到位。让浓缩胶聚合为1小时至过夜。 </li></ol> 4。存储凝胶<ol><li>当凝胶完全聚合，除去粘结剂剪辑，并放置在一个塑料袋热封的录像带，或重新密封的塑料袋。 </li><li>倒入20毫升1X叠加凝胶缓冲区包含入袋0.1％的叠氮化钠。 </li><li>热封袋为5秒，并确保有袋无泄漏。 </li><li>储存在4°C凝胶</li></ol> 5。样品准备<ol><li>在薄壁离心管中，加入6.5μL蛋白质样品，为7.5μl2X NOVEX Tris-甘氨酸SDS样品缓冲液，1μL100毫米二硫苏糖醇（DTT）和表意文字-H 2 O（如有必要）的总体积15μL。 </li><li>由涡旋混合样品，并短暂离心​​管底部带来内容的样本。 </li> &lt;李&gt;变性的样品在70°C为10分钟。 </li></ol> 6。运行凝胶<ol><li>成立NOVEX凝胶仪器。 </li><li>加入Tris-甘氨酸电泳缓冲液200毫升，包含500μL的内宅Nupage抗氧化剂，和300毫升的Tris-甘氨酸缓冲的外室。 </li><li>加载到使用凝胶加载枪头井的变性蛋白质样品（15μL）。 </li><li>运行60-70分钟，200至五凝胶。 </li></ol> 7。染色的凝胶<ol><li>从凝胶仪器取出录像带，并打破由两个塑料凝胶板，除了用胶刀或硬油灰刀撬环氧树脂密封。仔细分离板胶，不撕凝胶​​照顾。 </li><li>冲洗与表意文字- H凝胶2轻轻摇动3倍。 </li><li>污渍轻轻摇动1小时Invitrogen公司SimplyBlue SafeStain为凝胶。 </li><li>倒出染色液和脱色，凝胶与表意文字-H 2 O </li></ol> 8。清洁磁带<ol><li>使用金​​属铲刮或关闭所有磁带环氧树脂剥离，使他们能够再次被重用。 </li><li>每个塑料凝胶板用温和的洗涤剂溶液洗，自来水蒸馏水冲洗。 </li></ol> 9。代表结果因为环氧树脂会形成水密封，（图1），录音带不会泄漏时，丙烯酰胺溶液泼到他们。此外，凝胶录音带可以轻易打开凝胶染色检索，环氧树脂，可去皮的磁带，让他们继续再利用。正如图2所示，凝胶蛋白标记和样品带​​，适用于常规蛋白电泳显示清晰的分离。 <img src="/files/ftp_upload/3465/3465fig1.jpg" alt="图1" /> 图1。 APPLI阳离子环氧树脂密封微小胶体录音带。一个薄薄的一层环氧树脂应用到磁带的边缘装配前。为了突出环氧树脂，它是假色红色在这个形象。 <img src="/files/ftp_upload/3465/3465fig2.jpg" alt="图2" /> 图2。 SDS的Tris-甘氨酸凝胶的蛋白质分离浇微小胶体磁带重新使用SDS-PAGE进行如上所述。以下电泳凝胶取出沾满SimplyBlue SafeStain为制造商的指示，从微小胶体录像带，并拍下使用传送白光。巷中号包含SeeBlue Plus2预染蛋白标准。在车道中号的蛋白质的分子量道尔顿（kDa）的凝胶右侧上表示。其他车道​​显示清除从表达了谷胱甘肽-S-转移酶（GST）标签蛋白的细菌裂解的蛋白质。所示的蛋白质洗净，从Glutathione珠GST标签蛋白洗脱之前。

<ol>
	<li>Laemmli, U. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cleavage+of+structural+proteins+during+the+assembly+of+the+head+of+bacteriophage+T4.">Cleavage of structural proteins during the assembly of the head of bacteriophage T4.</a> Nature. 227, 680-685 (1970).</li><li>Raymond, S., Weintraub, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acrylamide+gel+as+a+supporting+medium+for+zone+electrophoresis.">Acrylamide gel as a supporting medium for zone electrophoresis.</a> Science. 130, 711-711 (1959).</li><li>Sch&#228;gger, H., von Jagow, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tricine-sodium+dodecyl+sulfate-polyacrylamide+gel+electrophoresis+for+the+separation+of+proteins+in+the+range+from+1+to+100+kDa.">Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa.</a> Anal. Biochem. 166, 368-379 (1987).</li><li>Shapiro, A. L., Vi&#241;uela, E., Maizel, J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+weight+estimation+of+polypeptide+chains+by+electrophoresis+in+SDS-polyacrylamide+gels.">Molecular weight estimation of polypeptide chains by electrophoresis in SDS-polyacrylamide gels.</a> Biochem. Biophys. Res. Commun. 28, 815-820 (1967).</li></ol>

没有利益冲突的声明。

蛋白质凝胶电泳是大多数分子生物学实验室不可或缺的方法。市售minigels提供了方便的较高水平，但可能是昂贵的。在这里，我们展示如何重新使用从的​​市售minigels的流行品牌的微小胶体录音带。此方法保留了方便，但有一​​个现成的凝胶源的市售minigels成本的一小部分。在此过程中的一个关键步骤是运用正确的金额环氧树脂完全密封录音带之前加入unpolymerized丙烯酰胺，以防止泄漏。只使用...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td> Invitrogen公司的Nupage的NOVEX微小胶体纸盒（1.0毫米） </td><td> Invitrogen公司</td><td> NC2010 </td><td>可在2，5，10，12，15和2D井</td></tr><tr><td>塑料环氧树脂</td><td>乐泰</td><td> 108771 </td><td></td></tr><tr><td>电工胶带</td><td>苏格兰威士忌</td><td></td><td></td></tr><tr><td> 125毫升侧臂瓶</td><td>耐热</td><td> 5360 </td><td></td></tr><tr><td> 4X Tris碱分离胶缓冲</td><td></td><td></td><td> 1.5 Tris碱的pH值8.7 </td></tr><tr><td>丙烯酰胺</td><td> Fisher Scientific则</td><td> 01065-500 </td><td></td></tr><tr><td> BIS-丙烯酰胺</td><td> MP Biomedicals， </td><td> 800173 </td><td></td></tr><tr><td> 30.8％的丙烯酰胺溶液</td><td></td><td></td><td> 30:0.8丙烯酰胺：双丙烯酰胺表意文字-H 2 O </td></tr><tr><td> 10％的APS </td><td> Fisher Scientific则</td><td> BP179-25 </td><td> 0.1克1毫升表意文字- H 2 O的过硫酸铵，立即使用前准备</td></tr><tr><td> TEMED </td><td> Arcos的有机物</td><td> 138450500 </td><td></td></tr><tr><td> 1 - 丁醇饱和1X分离胶缓冲</td><td></td><td></td><td> 5:2 1 - 丁醇1X分离胶缓冲</td></tr><tr><td> 4X Tris碱浓缩胶缓冲</td><td></td><td></td><td> 1.5 Tris碱pH值6.8 </td></tr><tr><td>凝胶梳</td><td> Invitrogen公司</td><td> NC3015 </td><td> 15好，1.0毫米厚度</td></tr><tr><td>脉冲封口机</td><td>趋势TEW电暖气设备</td><td></td><td></td></tr><tr><td>热封小包</td><td> kapak / Scotchpak </td><td></td><td> 1品脱大小（6.5“×8”） </td></tr><tr><td>凝胶存储缓冲区</td><td></td><td></td><td> 1X叠加0.1％叠氮化钠凝胶缓冲</td></tr><tr><td> NOVEX SDS样品缓冲液Tris-甘氨酸（2X） </td><td> Invitrogen公司</td><td> LC2676 </td><td></td></tr><tr><td>数码地面电视</td><td> FisherBiotech </td><td> BP172-5 </td><td></td></tr><tr><td> 10X的三Gylcine跑缓冲</td><td> Invitrogen公司</td><td> LC2675 </td><td> 10X股票1L：29.0Ğ三基地，144.0 g甘氨酸，10.0ĞSDS </td></tr><tr><td> Xcell的SureLock迷你细胞</td><td> Invitrogen公司</td><td> EI0001 </td><td></td></tr><tr><td>胶刀</td><td> Invitrogen公司</td><td> EI9010 </td><td></td></tr><tr><td> SimplyBlue安全染色</td><td> Invitrogen公司</td><td> LC6065 </tD&gt; <td></td></tr></tbody></table>

pouring and running a protein gel by reusing commercial cassettes

使用十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS - PAGE）分析蛋白质的评价是一种常见的技术，生物化学和分子生物学的研究人员1-4。执行每日蛋白质分析实验室，市售的聚丙烯酰胺凝胶（$ 10/gel）的成本可能是相当一段时间。为了降低成本，一些研究人员自己编写的聚丙烯酰胺凝胶。这些凝胶浇注的传统方法通常利用专门的设备和玻璃的凝胶板，可以是昂贵的，排除许多凝胶浇注和存储以供将来使用。此外，在清洗或浇注凝胶玻璃板处理可能会导致意外破损，创造一个安全隐患，这可能妨碍其使用在本科实验班。我们的协议演示如何同时倒入多个蛋白凝胶回收Invitrogen公司Nupage NOVEX微小胶体录音带，和廉价的马terials家装店购买。这种经济和精简的方法，包括了几个星期的方式储存在4°C凝胶。通过重新使用塑料胶盒从市售凝胶，实验室运行频繁的蛋白凝胶可显着节省成本，并帮助环境。此外，塑料胶录音带是极耐破损，这使得他们的本科生做实验教室的理想。

Watch this Scientific Journal Video about Pouring and Running a Protein Gel by reusing Commercial Cassettes at JoVE.com

Pouring and Running a Protein Gel by reusing Commercial Cassettes

我们的协议演示如何倒在时间的多种蛋白质凝胶回收Invitrogen公司Nupage NOVEX微小胶体录音带，并在家装商店购买廉价的材料。这种经济和精简的方法，包括了几个星期的方式储存在4°C凝胶。通过重新使用塑料胶盒从市售凝胶，实验室运行频繁的蛋白凝胶可显着节省成本，并帮助环境。

浇注和运行的一种蛋白质凝胶重用商​​业磁带

The evaluation of proteins using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis is a common technique used by biochemistry ...

pouring-and-running-a-protein-gel-by-reusing-commercial-cassettes

Research

JoVE Journal

Biology

24.8K 次观看.  University of Florida. 我们的协议演示如何倒在时间的多种蛋白质凝胶回收Invitrogen公司Nupage NOVEX微小胶体录音带，并在家装商店购买廉价的材料。这种经济和精简的方法，包括了几个星期的方式储存在4°C凝胶。通过重新使用塑料胶盒从市售凝胶，实验室运行频繁的蛋白凝胶可显着节省成本，并帮助环境。

视频: Pouring and Running a Protein Gel by reusing Commercial Cassettes

Screening for Amyloid Aggregation by Semi-Denaturing Detergent-Agarose Gel Electrophoresis

Amyloid aggregation is associated with numerous protein misfolding pathologies and underlies the infectious properties of prions, which are conformationally self-templating proteins that are thought to have beneficial roles in lower organisms.  Amyloids have been notoriously difficult to study due to their insolubility and structural heterogeneity.  However, resolution of amyloid polymers based on size and detergent insolubility has been made possible by Semi-Denaturing Detergent-Agarose Gel Electrophoresis (SDD-AGE).  This technique is finding widespread use for the detection and characterization of amyloid conformational variants.  Here, we demonstrate an adaptation of this technique that facilitates its use in large-scale applications, such as screens for novel prions and other amyloidogenic proteins.  The new SDD-AGE method uses capillary transfer for greater reliability and ease of use, and allows any sized gel to be accomodated.  Thus, a large number of samples, prepared from cells or purified proteins, can be processed simultaneously for the presence of SDS-insoluble conformers of tagged proteins.

SDD-AGE is a useful technique for the detection and characterization of amyloid-like polymers in cells. Here we demonstrate an adaptation that makes this technique amenable to large-scale applications.

半变性洗涤剂琼脂糖凝胶电泳筛选淀粉样蛋白聚合

Amyloid aggregation is associated with numerous protein misfolding pathologies and underlies the infectious properties of prions, which are ...

科研

生物学

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

无有机溶剂和醋酸，蛋白质与考马斯亮蓝G - 250凝胶染色

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based fractionation. The system is comprised of single-use, 8-sample capacity cartridges and a benchtop GELFREE Fractionation Instrument. During separation, a constant voltage is applied between the anode and cathode reservoirs, and each protein mixture is electrophoretically driven from a loading chamber into a specially designed gel column gel. Proteins are concentrated into a tight band in a stacking gel, and separated based on their respective electrophoretic mobilities in a resolving gel. As proteins elute from the column, they are trapped and concentrated in liquid phase in the collection chamber, free of the gel. The instrument is then paused at specific time intervals, and fractions are collected using a pipette. This process is repeated until all desired fractions have been collected. If fewer than 8 samples are run on a cartridge, any unused chambers can be used in subsequent separations.
This novel technology facilitates the quick and simple separation of up to 8 complex protein mixtures simultaneously, and offers several advantages when compared to previously available fractionation methods. This system is capable of fractionating up to 1mg of total protein per channel, for a total of 8mg per cartridge. Intact proteins over a broad mass range are separated on the basis of molecular weight, retaining important physiochemical properties of the analyte. The liquid phase entrapment provides for high recovery while eliminating the need for band or spot cutting, making the fractionation process highly reproducible1.

The accompanying video describes the use of the GELFREE 8100 Fractionation System, which partitions complex protein samples on the basis of molecular weight and recovers the fractions in liquid phase. The video describes how the technology works, how it is used, and provides resultant data, with polyacrylamide gel electrophoresis analysis of fractionated bovine liver homogenate.

液相恢复GELFREE 8100分馏系统使用基于分子量分馏

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based ...

Protein-protein Interactions Visualized by Bimolecular Fluorescence Complementation in Tobacco Protoplasts and Leaves

Many proteins interact transiently with other proteins or are integrated into multi-protein complexes to perform their biological function. Bimolecular fluorescence complementation (BiFC) is an in vivo method to monitor such interactions in plant cells. In the presented protocol the investigated candidate proteins are fused to complementary halves of fluorescent proteins and the respective constructs are introduced into plant cells via agrobacterium-mediated transformation. Subsequently, the proteins are transiently expressed in tobacco leaves and the restored fluorescent signals can be detected with a confocal laser scanning microscope in the intact cells. This allows not only visualization of the interaction itself, but also the subcellular localization of the protein complexes can be determined. For this purpose, marker genes containing a fluorescent tag can be coexpressed along with the BiFC constructs, thus visualizing cellular structures such as the endoplasmic reticulum, mitochondria, the Golgi apparatus or the plasma membrane. The fluorescent signal can be monitored either directly in epidermal leaf cells or in single protoplasts, which can be easily isolated from the transformed tobacco leaves. BiFC is ideally suited to study protein-protein interactions in their natural surroundings within the living cell. However, it has to be considered that the expression has to be driven by strong promoters and that the interaction partners are modified due to fusion of the relatively large fluorescence tags, which might interfere with the interaction mechanism. Nevertheless, BiFC is an excellent complementary approach to other commonly applied methods investigating protein-protein interactions, such as coimmunoprecipitation, in vitro pull-down assays or yeast-two-hybrid experiments.

Formation of protein complexes in vivo can be visualized by bimolecular fluorescence complementation. Interaction partners are fused to complementary parts of fluorescent tags and transiently expressed in tobacco leaves, resulting in a reconstituted fluorescent signal upon close proximity of the two proteins.

蛋白质 - 蛋白质相互作用的双分子荧光互补的烟草原生质体和叶片可视化

Many proteins interact transiently with other proteins or are integrated into multi-protein complexes to perform their biological function. Bimolecular ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

膜蛋白的绿色荧光蛋白为基础的表达筛选<em&gt;大肠杆菌</em

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, corticotrophin releasing hormone (CRH) is released from the hypothalamus in the brain. This stimulates the release of adrenocorticotrophic hormone (ACTH) from the pituitary gland, which in turn stimulates release of glucocorticoids from the adrenal gland. In horses the glucocorticoid corticosterone is responsible for several adaptations needed to support equine flight behaviour and subsequent removal from the aversive situation. Corticosterone metabolites can be detected in the feces of horses and assessment offers a non-invasive option to evaluate long term patterns of adrenal activity. Fecal assessment offers advantages over other techniques that monitor adrenal activity including blood plasma and saliva analysis. The non-invasive nature of the method avoids sampling stress which can confound results. It also allows the opportunity for repeated sampling over time and is ideal for studies in free ranging horses. This protocol describes the enzyme linked immunoassay (EIA) used to assess feces for corticosterone, in addition to the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. The method offers a non-invasive option to assess long term patterns in both domestic and free ranging horses. This protocol describes the enzyme linked immunoassay involved and the associated biochemical validation.

糖皮质激素粪分析：在马属动物无创监测肾上腺

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...

Detection of Glycosaminoglycans by Polyacrylamide Gel Electrophoresis and Silver Staining

Sulfated glycosaminoglycans (GAGs) such as heparan sulfate (HS) and chondroitin sulfate (CS) are ubiquitous in living organisms and play a critical role in a variety of basic biological structures and processes. As polymers, GAGs exist as a polydisperse mixture containing polysaccharide chains that can range from 4000 Da to well over 40,000 Da. Within these chains exists domains of sulfation, conferring a pattern of negative charge that facilitates interaction with positively charged residues of cognate protein ligands. Sulfated domains of GAGs must be of sufficient length to allow for these electrostatic interactions. To understand the function of GAGs in biological tissues, the investigator must be able to isolate, purify, and measure the size of GAGs. This report describes a practical and versatile polyacrylamide gel electrophoresis-based technique that can be leveraged to resolve relatively small differences in size between GAGs isolated from a variety of biological tissue types.

This report describes techniques to isolate and purify sulfated glycosaminoglycans (GAGs) from biological samples and a polyacrylamide gel electrophoresis approach to approximate their size. GAGs contribute to tissue structure and influence signaling processes via electrostatic interaction with proteins. GAG polymer length contributes to their binding affinity for cognate ligands.

聚丙烯酰胺凝胶电泳和银染色检测甘油糖

Sulfated glycosaminoglycans (GAGs) such as heparan sulfate (HS) and chondroitin sulfate (CS) are ubiquitous in living organisms and play a critical role ...

A Simple and Inexpensive Running Wheel Model for Progressive Resistance Training in Mice

Previously developed rodent resistance-based exercise models, including synergistic ablation, electrical stimulation, weighted-ladder climbing, and most recently, weighted-sled pulling, are highly effective at providing a hypertrophic stimulus to induce skeletal muscle adaptations. While these models have proven invaluable for skeletal muscle research, they are either invasive or involuntary and labor-intensive. Fortunately, many rodent strains voluntarily run long distances when given access to a running wheel. Loaded wheel running (LWR) models in rodents are capable of inducing adaptations commonly observed with resistance training in humans, such as increased muscle mass and fiber hypertrophy, as well as stimulation of muscle protein synthesis. However, the addition of moderate wheel load either fails to deter mice from running great distances, which is more reflective of an endurance/resistance training model, or the mice discontinue running nearly entirely due to the method of load application. Therefore, a novel high-load wheel running model (HLWR) has been developed for mice where external resistance is applied and progressively increased, enabling mice to continue running with much higher loads than previously utilized. Preliminary results from this novel HLWR model suggest it provides sufficient stimulus to induce hypertrophic adaptations over the 9 week training protocol. Herein, the specific procedures to execute this simple yet inexpensive progressive resistance-based exercise training model in mice are described.

This procedure describes a translatable progressive loaded running wheel resistance training model in mice. The primary advantage of this resistance training model is that it is entirely voluntary, thus reducing stress for the animals and the burden on the researcher.

一种简单且廉价的跑轮模型，用于小鼠的渐进式阻力训练

Previously developed rodent resistance-based exercise models, including synergistic ablation, electrical stimulation, weighted-ladder climbing, and most ...

使用改良基质凝胶塞测定简化血管生成研究

Modified In Vivo Matrix Gel Plug Assay for Angiogenesis Studies

Several models have been developed to investigate angiogenesis in vivo. However, most of these models are complex and expensive, require specialized equipment, or are hard to perform for subsequent quantitative analysis. Here we present a modified matrix gel plug assay to evaluate angiogenesis in vivo. In this protocol, vascular cells were mixed with matrix gel in the presence or absence of pro-angiogenic or anti-angiogenic reagents, and then subcutaneously injected into the back of recipient mice. After 7 days, phosphate buffer saline containing dextran-FITC is injected via the tail vein and circulated in vessels for 30 min. Matrix gel plugs are collected and embedded with tissue embedding gel, then 12 &#181;m sections are cut for fluorescence detection without staining. In this assay, dextran-FITC with high molecular weight (~150,000 Da) can be used to indicate functional vessels for detecting their length, while dextran-FITC with low molecular weight (~4,400 Da) can be used to indicate the permeability of neo-vessels. In conclusion, this protocol can provide a reliable and convenient method for the quantitative study of angiogenesis in vivo.

作者聚焦：通过改良基质凝胶塞测定研究血管生成和血管通透性

The method presented here can evaluate the effect of reagents on angiogenesis or vascular permeability in vivo without staining. The method uses dextran-FITC injection via the tail vein to visualize neo-vessels or vascular leakage.

用于血管生成研究 的改良体内 基质凝胶塞测定

Several models have been developed to investigate angiogenesis in vivo. However, most of these models are complex and expensive, require specialized ...

浇注和运行的一种蛋白质凝胶重用商​​业磁带

浇注和运行的一种蛋白质凝胶重用商业磁带