这项工作得到了拉里L ·希尔布鲁姆基金会，IIRG - 07 - 58334，从老年痴呆症协会，和来自加利福尼亚州公共卫生部07-65798和NIH / NIA的，2005/2E补助AG027818 AG030709。

1。肽制剂<ol><li>称取冻干肽100-200微克使用天平和转移到标记，涂硅，低吸附离心管。在这里，我们使用人类序列的Aβ40，Aβ42，降钙素和GRF。 </li><li>在这里，我们使用1,1,1,3,3,3 - 六氟-2 - 丙醇（异丙醇）预处理后，获得均匀，总无准备的肽。这一步是必要的，因为预先形成的集合体导致淀粉样蛋白的快速聚集，导致实验 5之间的重复性差。如过滤和美国证券交易委员会的其他方法也可以用于获取PICUP 6总的准备。 </li><li>为了治疗异丙醇肽，阴寒内通风穿着适当的保护罩（异丙醇挥发和有毒）冰异丙醇容器。一个250毫升的瓶子冷却通常需要10-15分钟。加入预冷管含有肽lyophilizates获得一个0.5毫米的名义肽浓度的异丙醇。 </li><li>超声肽的解决方案为在室温下5分钟的水浴sonicator。 </li><li>涡轻轻地在室温下30分钟的孵育管。 </li><li>冷藏冰（1分）管，并分为10 - 50 -μL分装在标有0.6毫升低吸附管的解决方案。 </li><li>删除下一个温柔的氮流蒸发异丙醇，或离开管在通风柜中打开过夜。一夜之间蒸发，放置在一个机架上的开放式管，并用大板，以防止灰尘和微粒污染的Kimwipes覆盖。 </li><li> Exsiccate在冷冻干燥机， 在真空中剩余的异丙醇或离心浓缩为30分钟，或在2小时真空入口exsiccator最终产品将在肽膜的离心管底部。如果正确exsiccated，可存储管（月）长时间密闭在-20或-80 ° C。 </li></ol> 2。溶异丙醇处理的肽和照片交联<ol><li>在溶异丙醇处理的肽交联反应，需要准备交联和淬火试剂。 </li><li>称取过硫酸铵（APS，议员228.2克/摩尔），并准备在10 mM磷酸钠，pH 7.4的20毫米的解决方案。混合使用一个旋涡，直到解决的办法是明确的。 </li><li>准备在10 mM磷酸钠，pH值7.4 1毫米的Tris溶液（2,2 - 联吡啶）dichlororuthenium（二）六水（RuBpy，议员748.63克/摩尔）。混合使用一个旋涡，并确认完全溶解。保护使用铝箔的光管。 </li><li> SDS - PAGE分析以下交联，一个方便的淬火试剂是5％β-巯基乙醇，在2 × SDS - PAGE样品缓冲液。另外，1米二硫苏糖醇（DTT，议员154.5克/摩尔）在去离子水或适当的缓冲区可以使用。 </li><li>异丙醇治疗肽片溶解于稀氢氧化钠第一和磷酸钠缓冲液，然后添加到〜30微米肽浓度。其次是去离子水含有肽电影等，氢氧化钠和水构成分别为10和45％的最终体积管加入60 mM的氢氧化钠。刮去使用离心管尖，向上和向下吹打混合，超声为5分钟在水浴sonicator内壁肽电影。 </li><li>添加45％20 mM磷酸钠，pH值7.4，由吹打混合，并在10分钟的16000克离心机。抛开uncross挂钩肽与阴性对照淬火试剂混合等份。 </li><li>调整相机的快门延迟为1秒。在较高的照射时间，大量的自由基反应可能会导致蛋白质的降解。装入相机的快门。照射时间可能需要优化时使用一个新的蛋白质的方法。 </li><li>一个典型的PICUP反应是在20μl的反应。转移到一个薄壁的，明确的，0.2毫升PCR管18μL肽的解决方案。 </li><li>肽的解决方案，添加1μLRuBpy 1μL的APS和混合试剂移液器（RuBpy和APS终浓度在反应混合物将分别为0.05和1毫米，）。 </li><li>快速放置在反应管内的1.8 mL的玻璃小瓶。放置在相机机身的前面附加波纹管内的小瓶。将镜头保护盖和按快门，使样品照射1波纹管内的小号。 </li><li>样品照射后，迅速采取波纹管和PCR管瓶小瓶中。快速淬火，加入1μL的DTT或10μL减少PAGE样品缓冲液的反应。重复每个肽等分的反应。 </li><li>反应混合物现在可以不再超过7天，或放在冰上SDS - PAGE电泳和银染分析前冷冻储存在-20 ° C。 </li></ol> 3。 SDS - PAGE和银染色交联肽产品<ol><li>常规SDS - PAGE电泳和银染进行可视化交联肽。 </li><li>装入5μL反应混合物中的每通道获得每巷肽〜130 pmol。 </li><li>包括比较类似的uncross相连的肽。还包括一个标准蛋白分子量肽乐队的视觉逼近的阶梯。 </li><li>运行使用一个标准的凝胶电泳仪凝胶。我们使用自Invitrogen XCell SureLock小型电池系统，并按照Invitrogen公司出版IM - 1002，NOVEX预制凝胶电泳指南进行银染。 </li></ol>第4部分：代表性的结果（图1） <img src="/files/ftp_upload/old/1071_2009_1_19_13/Result no demarcation.png" alt="图1" /> 图1：银染聚丙烯酰胺凝胶电泳显示uncross挂钩（ - ）及光交联（+），GRF，降钙素，Aβ40和Aβ42 SDS - PAGE和银染分析显示PICUP产生的Aβ40低聚物单体大致相同，通过四聚体带的强度，其次是一个在更高的低聚物7丰度的急剧下降。 Uncross联系Aβ40迁移与先生与单体7一致。 Aβ42显示了独特的低聚物的粒度分布。 Aβ42的低聚物包括2-3组8。第一组，通过三聚体单体，显示越来越低聚物秩序的力度递减。在第二组中，高斯状分布，是观察之间的四聚体，heptamer，最多五聚体和六聚体。第三组，这是这里没有显示，包含先生〜8 30,000-60,000大的低聚物。这些较高的议员，低聚物通常使用Aβ42的低分子量SEC隔离观察，但不肽准备通过过滤或异丙醇治疗 6 。 Uncross挂钩Aβ42的产生主要是两个频段，单体带和广阔的三聚体/四聚体带，这是一个人工制品经SDS 5,9诱导。降钙素产量低聚物的大小分布，强烈发散通过四聚体的地区单体单调指数下降，表明预存在，二聚体，三聚体和四聚体7。控制多肽，GRF，产生一种单调的低聚物分布范围从二聚体通过六聚体等，明显的相对含量低聚物分子质量的增加呈指数下降。在其他实验中，较高的低聚物也可以可视化7。确切的数字和低聚物带的相对强度实验是根据实际的蛋白浓度，凝胶上装载的蛋白质的量，并在银染协议的开发步骤中所用的时间可能会有所不同。

<ol>
	<li>Bitan, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+study+of+metastable+amyloidogenic+protein+oligomers+by+photo-induced+cross-linking+of+unmodified+proteins.">Structural study of metastable amyloidogenic protein oligomers by photo-induced cross-linking of unmodified proteins.</a> Methods Enzymol. 413, 217-236 (2006).</li><li>Fancy, D. A., Kodadek, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemistry+for+the+analysis+of+protein-protein+interactions:+rapid+and+efficient+cross-linking+triggered+by+long+wavelength+light.">Chemistry for the analysis of protein-protein interactions: rapid and efficient cross-linking triggered by long wavelength light.</a> Proc. Natl. Acad. Sci. 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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amyloid+&#946;-protein+oligomerization:+prenucleation+interactions+revealed+by+photo-induced+cross-linking+of+unmodified+proteins.">Amyloid &#946;-protein oligomerization: prenucleation interactions revealed by photo-induced cross-linking of unmodified proteins.</a> J. Biol. Chem. 276, 35176-35184 (2001).</li><li>Bitan, G., Kirkitadze, M. D., Lomakin, A., Vollers, S. S., Benedek, G. B., Teplow, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amyloid+&#946;-protein+(A&#946;)+assembly:+A&#946;40+and+A&#946;42+oligomerize+through+distinct+pathways.">Amyloid &#946;-protein (A&#946;) assembly: A&#946;40 and A&#946;42 oligomerize through distinct pathways.</a> Proc. Natl. Acad. Sci. USA. 100, 330-335 (2003).</li><li>Hepler, R. W., Grimm, K. M., Nahas, D. 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PICUP最初开发研究稳定的蛋白复合物 2 。该方法应用定量研究亚淀粉样蛋白的组件，包括Aβ 的 10朊病毒疾病相关的PrP SC 11和α-突触核蛋白 12 。 PICUP实验设计时必须考虑的最重要的因素是试剂的化学计量学，激光照射时间，和样品制备过程。前两个问题，可能需要实证的优化，而后者的问题在很大程度上影响实验数据的解释。特别是对于淀粉样蛋白，亚稳低聚物的粒度分布的测定...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Dolan-Jenner 200-W incandescent lamp</td>
<td>Other</td>
<td>Dolan-Jenner Industries</td>
<td>Model 170-D</td>
<td>The heat generated by the lamp does not affect samples for short incubation periods.</td>
</tr>
<tr>
<td>35-mm SLR camera body</td>
<td>Tool</td>
<td>Pentax</td>
<td>SP500 model</td>
<td>In our settings, a bellows is attached to the body of the camera to provide a convenient chamber for irradiation of the sample placed 10 cm away from the light source.</td>
</tr>
<tr>
<td>Clear, thin walled PCR tubes</td>
<td>Other</td>
<td>Eppendorf</td>
<td>951010006 supplied by Fisher L22-003-24</td>
<td>&nbsp;</td>
<tr>
<td>Glass vials (1.8 mL)</td>
<td>Other</td>
<td>Kimble Chromatography</td>
<td>60940A 2, supplied by Fisher 03-340-60</td>
<td>&nbsp;</td>
<tr>
<td>GRF</td>
<td>Reagent</td>
<td>Bachem</td>
<td>H-3695</td>
<td>&nbsp;</td>
<tr>
<td>HFIP</td>
<td>Reagent</td>
<td>TCI America</td>
<td>H0424</td>
<td>Use in a fume hood.</td>
</tr>
<tr>
<td>A&beta;40 and A&beta;42</td>
<td>Reagent</td>
<td>UCLA Biopolymers Laboratory</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Calcitonin</td>
<td>Reagent</td>
<td>American Peptide</td>
<td>22-1-10</td>
<td>&nbsp;</td>
<tr>
<td>Tris(2,2-bipridyl)dichlororuthenium(II) hexahydrate</td>
<td>Reagent</td>
<td>Sigma</td>
<td>224758-1G</td>
<td>Vortex until the solution is clear. Cover the RuBpy tube with foil to protect the reagent from ambient light. RuBpy is prepared freshly each time and should be used within 48 h.</td>
</tr>
<tr>
<td>Ammonium persulfate</td>
<td>Reagent</td>
<td>Sigma</td>
<td>A-7460</td>
<td>Vortex until the solution is clear. APS is prepared freshly each time and should be used within 48 h. </td>
</tr>
<tr>
<td>&beta;-mercaptoethanol</td>
<td>Reagent</td>
<td>Sigma</td>
<td>M7154-25 ML</td>
<td>Can be used when SDS-PAGE analysis is performed.</td>
</tr>
<tr>
<td>Novex Tricine SDS Sample Buffer (2x)</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>LC1676</td>
<td>&nbsp;</td>
<tr>
<td>XCell SureLock Mini-Cell</td>
<td>Tool</td>
<td>Invitrogen</td>
<td>EI0001</td>
<td>&nbsp;</td>
<tr>
<td>Novex Tricine Gels (10-20%)</td>
<td>Other</td>
<td>Invitrogen</td>
<td>EC6625B0X</td>
<td>&nbsp;</td>
<tr>
<td>Novex Tricine SDS Running Buffer (10x )</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>LC1675</td>
<td>&nbsp;</td>
<tr>
<td>Silver Express Staining Kit</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>LC6100</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

photo-induced cross-linking of unmodified proteins (picup) applied to amyloidogenic peptides

淀粉样蛋白装配成有毒的低聚物是蛋白质错误折叠疾病，包括阿尔茨海默氏症，帕金森氏症，亨廷顿氏病，遗传性肌萎缩性脊髓侧索硬化症，与2型糖尿病的发病中的一个开创性的事件。由于这些蛋白质组件的亚稳的性质，它是很难用传统的方法，如电泳，色谱仪，荧光，或动态光散射，定量，以评估他们的齐聚物的粒度分布。淀粉样蛋白的低聚物存在亚稳态的混合物，其中的低聚物分解成单体，同时关联到更大的集会。 PICUP稳定的共价交联PICUP时，分馏的方法，如十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS - PAGE）或体积排阻色谱法（SEC），相结合，提供快照齐聚物的粒度分布，前交叉存在的低聚物人口联。因此，PICUP使亚稳态蛋白人群的可视化和定量分析，可用于监控大会和破译关系之间的序列修改和齐聚<sup&gt; 1</sup&gt;。机械，PICUP涉及光氧化钌<sup&gt; 2 +</sup在一个三（联吡啶）钌（Ⅱ）配合物（RuBpy）茹<sup&gt; 3 +</sup&gt;与电子受体的存在可见光照射。茹<sup&gt; 3 +</sup&gt;是一个强大的电子氧化剂，能够抽象从邻近的蛋白质分子的电子，自由基生成一种蛋白质<sup&gt; 1,2</sup&gt;。自由基是不稳定的，高活性物种，因此，通过各种内和分子间的反应迅速消失。一个激进的可能会利用高能量的未成对电子的反应与另一种蛋白质的形成一个二聚体自由基，随后失去一个氢原子，并形成了一个稳定的，共价键相连的二聚体的单体。通过类似的机制，以形成高阶低聚物与单体或其他二聚体，然后二聚体可进一步反应。 PICUP相对其他照片或化学交联方法的优点<sup&gt; 3,4</sup&gt;包括短期非破坏性的可见光（≤1秒）的曝光，没有必要<i前事实</i&gt;修改本机的序列和长度为零的共价交联。此外，PICUP使交联蛋白在广泛的pH值和温度范围内，包括生理参数。在这里，我们展示了三个淀粉样蛋白40交叉连接 - 和42个氨基酸残基的淀粉样β-蛋白变体（Aβ40和Aβ42的），和降钙素PICUP应用程序，以及控制蛋白，生长激素释放因子（GRF）。

Watch this Scientific Journal Video about Photo-Induced Cross-Linking of Unmodified Proteins PICUP Applied to Amyloidogenic Peptides at JoVE.com

Photo-Induced Cross-Linking of Unmodified Proteins PICUP Applied to Amyloidogenic Peptides

光致交联未修改的蛋白质（PICUP）允许低聚物在亚稳态的蛋白质混合物的粒度分布的特性。我们展示PICUP应用程序的三个有代表性的淀粉样肽的40 - 42残留的淀粉样β-蛋白和降钙素形式，控制肽生长激素释放因子。

光致交联未修改蛋白（PICUP）适用于淀粉样肽

The assembly of amyloidogenic proteins into toxic oligomers is a seminal event in the pathogenesis of protein misfolding diseases, including Alzheimer's, ...

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JoVE Journal

Biology

Photo-Induced Cross-Linking of Unmodified Proteins (PICUP) Applied to Amyloidogenic Peptides

21.2K 次观看.  University of California, Los Angeles. 光致交联未修改的蛋白质（PICUP）允许低聚物在亚稳态的蛋白质混合物的粒度分布的特性。我们展示PICUP应用程序的三个有代表性的淀粉样肽的40 - 42残留的淀粉样β-蛋白和降钙素形式，控制肽生长激素释放因子。

视频: Photo-Induced Cross-Linking of Unmodified Proteins PICUP Applied to Amyloidogenic Peptides

Electrophoretic Separation of Proteins

Electrophoresis is used to separate complex mixtures of proteins (e.g., from cells, subcellular fractions, column fractions, or immunoprecipitates), to investigate subunit compositions, and to verify homogeneity of protein samples. It can also serve to purify proteins for use in further applications. In polyacrylamide gel electrophoresis, proteins migrate in response to an electrical field through pores in a polyacrylamide gel matrix; pore size decreases with increasing acrylamide concentration. The combination of pore size and protein charge, size, and shape determines the migration rate of the protein. In this unit, the standard Laemmli method is described for discontinuous gel electrophoresis under denaturing conditions, i.e., in the presence of sodium dodecyl sulfate (SDS). 

In this video, we demonstrate a method for electrophoretic separation of proteins using poly-acrylimide gel electrophoresis (PAGE).

电泳分离蛋白质

Electrophoresis is used to separate complex mixtures of proteins (e.g., from cells, subcellular fractions, column fractions, or immunoprecipitates), to ...

科研

生物学

Staining Proteins in Gels

Following separation by electrophoretic methods, proteins in a gel can be detected by several staining methods. This unit describes protocols for detecting proteins by four popular methods. Coomassie blue staining is an easy and rapid method. Silver staining, while more time consuming, is considerably more sensitive and can thus be used to detect smaller amounts of protein. Fluorescent staining is a popular alternative to traditional staining procedures, mainly because it is more sensitive than Coomassie staining, and is often as sensitive as silver staining. Staining of proteins with SYPRO Orange and SYPRO Ruby are also demonstrated here.

Following separation by electrophoretic methods, proteins in a gel can be detected by several staining methods. Staining of proteins with Coomassie Blue, Silver Staining, SYPRO Orange, SYPRO Ruby are demonstrated in this video.

在凝胶染色蛋白质

Following separation by electrophoretic methods, proteins in a gel can be detected by several staining methods. This unit describes protocols for ...

A Lectin HPLC Method to Enrich Selectively-glycosylated Peptides from Complex Biological Samples

Glycans are an important class of post-translational modifications. Typically found on secreted and extracellular molecules, glycan structures signal the internal status of the cell. Glycans on tumor cells tend to have abundant sialic acid and fucose moieties. We propose that these cancer-associated glycan variants be exploited for biomarker development aimed at diagnosing early-stage disease. Accordingly, we developed a mass spectrometry-based workflow that incorporates chromatography on affinity matrices formed from lectins, proteins that bind specific glycan structures. The lectins Sambucus nigra (SNA) and Aleuria aurantia (AAL), which bind sialic acid and fucose, respectively, were covalently coupled to POROS beads (Applied Biosystems) and packed into PEEK columns for high pressure liquid chromatography (HPLC). Briefly, plasma was depleted of the fourteen most abundant proteins using a multiple affinity removal system (MARS-14; Agilent). Depleted plasma was trypsin-digested and separated into flow-through and bound fractions by SNA or AAL HPLC. The fractions were treated with PNGaseF to remove N-linked glycans, and analyzed by LC-MS/MS on a QStar Elite. Data were analyzed using Mascot software. The experimental design included positive controls&mdash;fucosylated and sialylated human lactoferrin glycopeptides&mdash;and negative controls&mdash;high mannose glycopeptides from Saccharomyces cerevisiae&mdash;that were used to monitor the specificity of lectin capture. Key features of this workflow include the reproducibility derived from the HPLC format, the positive identification of the captured and PNGaseF-treated glycopeptides from their deamidated Asn-Xxx-Ser/Thr motifs, and quality assessment using glycoprotein standards. Protocol optimization also included determining the appropriate ratio of starting material to column capacity, identifying the most efficient capture and elution buffers, and monitoring the PNGaseF-treatment to ensure full deglycosylation. Future directions include using this workflow to perform mass spectrometry-based discovery experiments on plasma from breast cancer patients and control individuals.

Lectin-conjugated POROS beads were employed for HPLC. Glycopeptide standards served as positive and negative controls. MARS-14 depleted, trypsin-digested human plasma was chromatographed and flow-through (FT) and bound fractions collected for ESI-LC-MS/MS analyses. Glycopeptides were enriched in the bound fraction as compared to FT.

一个外源凝集素的高效液相色谱法从复杂生物样品，以丰富的选择性糖基化肽

Glycans are an important class of post-translational modifications. Typically found on secreted and extracellular molecules, glycan structures signal the ...

Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

The dynamic nature of cellular machineries is frequently built on transient and/or weak protein associations. These low affinity interactions preclude stringent methods for the isolation and identification of protein networks around a protein of interest. The use of chemical crosslinkers allows the selective stabilization of labile interactions, thus bypassing biochemical limitations for purification. Here we present a protocol amenable for cells in culture that uses a homobifunctional crosslinker with a spacer arm of 12 &Aring;, dithiobis-(succinimidyl proprionate) (DSP). DSP is cleaved by reduction of a disulphide bond present in the molecule. Cross-linking combined with immunoaffinity chromatography of proteins of interest with magnetic beads allows the isolation of protein complexes that otherwise would not withstand purification. This protocol is compatible with regular western blot techniques and it can be scaled up for protein identification by mass spectrometry1.
Stephanie A. Zlatic and Pearl V. Ryder contributed equally to this work.

Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

The cell permeable crosslinker DSP [dithiobis-(succinimidyl propionate)] stabilizes transient and labile interactions in vivo, which allows their isolation using stringent protein complex purification techniques. Here we present a technique for crosslinking cells grown in culture followed by isolation of protein complexes by immunoprecipitation.

由不稳定的多蛋白质复合物的分离在体内控制细胞的交联和免疫磁性亲和层析

The dynamic nature of cellular machineries is frequently built on transient and/or weak protein associations. These low affinity interactions preclude ...

Laser Microdissection Applied to Gene Expression Profiling of Subset of Cells from the Drosophila Wing Disc

Heterogeneous nature of tissues has proven to be a limiting factor in the amount of information that can be generated from biological samples, compromising downstream analyses. Considering the complex and dynamic cellular associations existing within many tissues, in order to recapitulate the in vivo interactions thorough molecular analysis one must be able to analyze specific cell populations within their native context. Laser-mediated microdissection can achieve this goal, allowing unambiguous identification and successful harvest of cells of interest under direct microscopic visualization while maintaining molecular integrity. We have applied this technology to analyse gene expression within defined areas of the developing Drosophila wing disc, which represents an advantageous model system to study growth control, cell differentiation and organogenesis. Larval imaginal discs are precociously subdivided into anterior and posterior, dorsal and ventral compartments by lineage restriction boundaries. Making use of the inducible GAL4-UAS binary expression system, each of these compartments can be specifically labelled in transgenic flies expressing an UAS-GFP transgene under the control of the appropriate GAL4-driver construct. In the transgenic discs, gene expression profiling of discrete subsets of cells can precisely be determined after laser-mediated microdissection, using the fluorescent GFP signal to guide laser cut. 
Among the variety of downstream applications, we focused on RNA transcript profiling after localised RNA interference (RNAi). With the advent of RNAi technology, GFP labelling can be coupled with localised knockdown of a given gene, allowing to determinate the transcriptional response of a discrete cell population to the specific gene silencing. To validate this approach, we dissected equivalent areas of the disc from the posterior (labelled by GFP expression), and the anterior (unlabelled) compartment upon regional silencing in the P compartment of an otherwise ubiquitously expressed gene. RNA was extracted from microdissected silenced and unsilenced areas and comparative gene expression profiling determined by quantitative real-time RT-PCR. We show that this method can effectively be applied for accurate transcriptomics of subsets of cells within the Drosophila imaginal discs. Indeed, while massive disc preparation as source of RNA generally assumes cell homogeneity, it is well known that transcriptional expression can vary greatly within these structures in consequence of positional information. Using localized fluorescent GFP signal to guide laser cut, more accurate transcriptional analyses can be performed and profitably applied to disparate applications, including transcript profiling of distinct cell lineages within their native context.

Laser Microdissection Applied to Gene Expression Profiling of Subset of Cells from the Drosophila Wing Disc

Laser microdissection was applied to analyse gene expression profiling in specific compartments of Drosophila wing disc subjected to localised RNAi in vivo. RNA extracted from equivalent areas of silenced and unsilenced compartments was analysed by quantitative RT-PCR to determine comparative gene expression profiling within the context of native tissue microecology.

激光显微切割的从细胞的一个子集基因表达分析中的应用果蝇永光盘

Heterogeneous nature of tissues has proven to be a limiting factor in the amount of information that can be generated from biological samples, ...

High Throughput Quantitative Expression Screening and Purification Applied to Recombinant Disulfide-rich Venom Proteins Produced in E. coli

Escherichia coli (E. coli) is the most widely used expression system for the production of recombinant proteins for structural and functional studies. However, purifying proteins is sometimes challenging since many proteins are expressed in an insoluble form. When working with difficult or multiple targets it is therefore recommended to use high throughput (HTP) protein expression screening on a small scale (1-4&#160;ml cultures) to quickly identify conditions for soluble expression. To cope with the various structural genomics programs of the lab, a quantitative (within a range of 0.1-100 mg/L culture of recombinant protein) and HTP protein expression screening protocol was implemented and validated on thousands of proteins. The protocols were automated with the use of a liquid handling robot but can also be performed manually without specialized equipment.
Disulfide-rich venom proteins are gaining increasing recognition for their potential as therapeutic drug leads. They can be highly potent and selective, but their complex disulfide bond networks make them challenging to produce. As a member of the FP7 European Venomics project (<a href="http://www.venomics.eu">www.venomics.eu</a>), our challenge is to develop successful production strategies with the aim of producing thousands of novel venom proteins for functional characterization. Aided by the redox properties of disulfide bond isomerase DsbC, we adapted our HTP production pipeline for the expression of oxidized, functional venom peptides in the E. coli cytoplasm. The protocols are also applicable to the production of diverse disulfide-rich proteins. Here we demonstrate our pipeline applied to the production of animal venom proteins. With the protocols described herein it is likely that soluble disulfide-rich proteins will be obtained in as little as a week. Even from a small scale, there is the potential to use the purified proteins for validating the oxidation state by mass spectrometry, for characterization in pilot studies, or for sensitive micro-assays.

High Throughput Quantitative Expression Screening and Purification Applied to Recombinant Disulfide-rich Venom Proteins Produced in E. coli

A protocol for the quantitative, high throughput expression screening and analytical purification of fusion proteins from small-scale Escherichia coli cultures is described and applied to the expression of disulfide-rich animal venom protein targets.

高通量定量表达筛选与纯化应用到重组二硫键丰富的蛇毒蛋白产于<em&gt; E。大肠杆菌</em

Escherichia coli (E. coli) is the most widely used expression system for the production of recombinant proteins for structural and functional studies. ...

Non-chromatographic Purification of Recombinant Elastin-like Polypeptides and their Fusions with Peptides and Proteins from Escherichia coli

Elastin-like polypeptides are repetitive biopolymers that exhibit a lower critical solution temperature phase transition behavior, existing as soluble unimers below a characteristic transition temperature and aggregating into micron-scale coacervates above their transition temperature. The design of elastin-like polypeptides at the genetic level permits precise control of their sequence and length, which dictates their thermal properties. Elastin-like polypeptides are used in a variety of applications including biosensing, tissue engineering, and drug delivery, where the transition temperature and biopolymer architecture of the ELP can be tuned for the specific application of interest. Furthermore, the lower critical solution temperature phase transition behavior of elastin-like polypeptides allows their purification by their thermal response, such that their selective coacervation and resolubilization allows the removal of both soluble and insoluble contaminants following expression in Escherichia coli. This approach can be used for the purification of elastin-like polypeptides alone or as a purification tool for peptide or protein fusions where recombinant peptides or proteins genetically appended to elastin-like polypeptide tags can be purified without chromatography. This protocol describes the purification of elastin-like polypeptides and their peptide or protein fusions and discusses basic characterization techniques to assess the thermal behavior of pure elastin-like polypeptide products.

Non-chromatographic Purification of Recombinant Elastin-like Polypeptides and their Fusions with Peptides and Proteins from Escherichia coli

Elastin-like polypeptides are stimulus-responsive biopolymers with applications ranging from recombinant protein purification to drug delivery. This protocol describes the purification and characterization of elastin-like polypeptides and their peptide or protein fusions from Escherichia coli using their lower critical solution temperature phase transition behavior as a simple alternative to chromatography.

重组弹性蛋白样多肽及其融合物与从肽和蛋白质非色谱纯化<em&gt;大肠杆菌</em

Elastin-like polypeptides are repetitive biopolymers that exhibit a lower critical solution temperature phase transition behavior, existing as soluble ...

Use of MALDI-TOF Mass Spectrometry and a Custom Database to Characterize Bacteria Indigenous to a Unique Cave Environment (Kartchner Caverns, AZ, USA)

MALDI-TOF mass spectrometry has been shown to be a rapid and reliable tool for identification of bacteria at the genus and species, and in some cases, strain levels. Commercially available and open source software tools have been developed to facilitate identification; however, no universal/standardized data analysis pipeline has been described in the literature. Here, we provide a comprehensive and detailed demonstration of bacterial identification procedures using a MALDI-TOF mass spectrometer. Mass spectra were collected from 15 diverse bacteria isolated from Kartchner Caverns, AZ, USA, and identified by 16S rDNA sequencing. Databases were constructed in BioNumerics 7.1. Follow-up analyses of mass spectra were performed, including cluster analyses, peak matching, and statistical analyses. Identification was performed using blind-coded samples randomly selected from these 15 bacteria. Two identification methods are presented: similarity coefficient-based and biomarker-based methods. Results show that both identification methods can identify the bacteria to the species level.

Use of MALDI-TOF Mass Spectrometry and a Custom Database to Characterize Bacteria Indigenous to a Unique Cave Environment Kartchner Caverns, AZ, USA

This work details procedures for rapid identification of bacteria using MALDI-TOF MS. The identification procedures include spectrum acquisition, database construction, and follow up analyses. Two identification methods, similarity coefficient-based and biomarker-based methods, are presented.

使用MALDI-TOF质谱和自定义数据库的表征细菌土著一个独特的洞穴环境（卡切内岩洞，AZ，USA）

MALDI-TOF mass spectrometry has been shown to be a rapid and reliable tool for identification of bacteria at the genus and species, and in some cases, ...

A Method for Selecting Structure-switching Aptamers Applied to a Colorimetric Gold Nanoparticle Assay

Small molecules provide rich targets for biosensing applications due to their physiological implications as biomarkers of various aspects of human health and performance. Nucleic acid aptamers have been increasingly applied as recognition elements on biosensor platforms, but selecting aptamers toward small molecule targets requires special design considerations. This work describes modification and critical steps of a method designed to select structure-switching aptamers to small molecule targets. Binding sequences from a DNA library hybridized to complementary DNA capture probes on magnetic beads are separated from nonbinders via a target-induced change in conformation. This method is advantageous because sequences binding the support matrix (beads) will not be further amplified, and it does not require immobilization of the target molecule. However, the melting temperature of the capture probe and library is kept at or slightly above RT, such that sequences that dehybridize based on thermodynamics will also be present in the supernatant solution. This effectively limits the partitioning efficiency (ability to separate target binding sequences from nonbinders), and therefore many selection rounds will be required to remove background sequences. The reported method differs from previous structure-switching aptamer selections due to implementation of negative selection steps, simplified enrichment monitoring, and extension of the length of the capture probe following selection enrichment to provide enhanced stringency. The selected structure-switching aptamers are advantageous in a gold nanoparticle assay platform that reports the presence of a target molecule by the conformational change of the aptamer. The gold nanoparticle assay was applied because it provides a simple, rapid colorimetric readout that is beneficial in a clinical or deployed environment. Design and optimization considerations are presented for the assay as proof-of-principle work in buffer to provide a foundation for further extension of the work toward small molecule biosensing in physiological fluids.

A protocol is provided to select structure-switching aptamers for small molecule targets based on a tunable stringency magnetic bead selection method. Aptamers selected with structure-switching properties are beneficial for assays that require a conformational change to signal the presence of a target, such as the described gold nanoparticle assay.

适用于比色金纳米粒子检测方法的选择结构切换适配方法

Small molecules provide rich targets for biosensing applications due to their physiological implications as biomarkers of various aspects of human health ...

Enrichment of Extracellular Matrix Proteins from Tissues and Digestion into Peptides for Mass Spectrometry Analysis

The extracellular matrix (ECM) is a complex meshwork of cross-linked proteins that provides biophysical and biochemical cues that are major regulators of cell proliferation, survival, migration, etc. The ECM plays important roles in development and in diverse pathologies including cardio-vascular and musculo-skeletal diseases, fibrosis, and cancer. Thus, characterizing the composition of ECMs of normal and diseased tissues could lead to the identification of novel prognostic and diagnostic biomarkers and potential novel therapeutic targets. However, the very nature of ECM proteins (large in size, cross-linked and covalently bound, heavily glycosylated) has rendered biochemical analyses of ECMs challenging. To overcome this challenge, we developed a method to enrich ECMs from fresh or frozen tissues and tumors that takes advantage of the insolubility of ECM proteins. We describe here in detail the decellularization procedure that consists of sequential incubations in buffers of different pH and salt and detergent concentrations and that results in 1) the extraction of intracellular (cytosolic, nuclear, membrane and cytoskeletal) proteins and 2) the enrichment of ECM proteins. We then describe how to deglycosylate and digest ECM-enriched protein preparations into peptides for subsequent analysis by mass spectrometry.

This protocol describes a procedure for enriching ECM proteins from tissues or tumors and deglycosylating and digesting the ECM-enriched preparations into peptides to analyze their protein composition by mass spectrometry.

从富集组织及消化细胞外基质蛋白的成肽质谱分析

The extracellular matrix (ECM) is a complex meshwork of cross-linked proteins that provides biophysical and biochemical cues that are major regulators of ...