争取视线和研究防盲提供了资金。

1。眼前段解剖。 <ol><li>角膜缘后巩膜组织掌握0.22 forcep和全球（眼球）是稳定的。 </li><li>一个显微刀片是用来在从角膜缘角膜缘的角膜线性切口。 </li><li>然后用0.12科利柏forcep把握角膜切口。 </li><li>前房液，然后吸收与Weck - CEL手术矛。 </li></ol> 2。镜头剜。 <ol><li> 0.12科利柏forcep抓角膜用于稳定全球。 </li><li>一个良好的弯针持有人（或弯曲敷料钳）插入后面的镜头走向全球的后路方面。持针器是局部封闭，并向前拉。巩膜，推动通过角膜切口的镜头而离开眼球壁的完整性的外部表面上使用镊子施加压力。玻璃体出现一个半透明的凝胶，部分附着在镜头上。 </li><li>镜头玻璃体组织，然后放入含有20微升PBS溶解蛋白酶抑制剂的鸡尾酒（罗氏）一个过滤离心管。 </li></ol> 3。视网膜剜。 <ol><li>继续稳定与0.12科利柏抓角膜切口钳全球。 </li><li>一个良好的弯针持有人（或弯曲敷料钳）是放在全球尽可能远后，附近的视神经。持针器是局部封闭，并向前拉。巩膜，视网膜推动通过角膜切口的外部表面上使用镊子施加压力。作为一个半透明的凝胶，附着于视网膜玻璃体出现。视网膜是为黄色，吻合血管的组织的可视化。色素组织的任何条可用镊子剥离。 </li><li>玻璃体视网膜组织，然后放入过滤离心机包含镜头玻璃体组织管。 </li></ol> 4。过滤的离心。 <ol><li>过滤离心管放置在台式离心机。 14000 X克分拆为12分钟。 </li><li>从下腔，这是玻璃体吸洗脱。视网膜仍然在上议院。这些样本可以用于蛋白质研究。 </li></ol> 5。代表性的成果经SDS - PAGE（图2）玻璃体样本进行了分析。样品也被用于酶活性测定（图3）。 <img src="/files/ftp_upload/2795/2795fig1.jpg" alt="图1"/> 图1。老鼠眼图。横断面示意图显示了人类和小鼠的眼睛中由于其较大的镜头鼠标眼睛玻璃体体积相对较小。 <img src="/files/ftp_upload/2795/2795fig2.jpg" alt="图2"/> 图2一维的SDS - PAGE鼠标玻璃体和视网膜。鼠标玻璃体蛋白质图谱，13.6毫克/毫升（1巷），和视网膜，11.35毫克/毫升（2巷）。凝胶电泳在150千伏为45分钟，使用Flamingo荧光凝胶染色的染色和可视化使用VersaDoc成像系统（BioRad公司，大力士，CA）。 <img src="/files/ftp_upload/2795/2795fig3.jpg" alt="图3"/> 图3。超氧化物歧化酶（SOD）酶的活性在小鼠和人的玻璃体 。剜DBA和白化小鼠收集的玻璃体和视网膜上执行测量总SOD活性（SOD活性比色法aasay套件，＃AB65354，Abcam公司，公司，剑桥，马萨诸塞）。这是相比，在人类玻璃体核心和人类从验尸报告的人类捐助眼睛的视网膜收集到的确定的相对活性SOD活性。手动吸气，用一个23号针头，11日和开花的眼睛和切割组织，远离复杂的RPE /脉络膜视网膜收集，未经稀释的玻璃体核心样品。虽然检测不区分超氧化物歧化酶异构体，玻璃体SOD活性很可能反映SOD3，因为它是唯一的外亚型 12视网膜SOD活性很可能反映了所有三个超氧化物歧化酶亚型：是细胞内的SOD1，线粒体SOD2 ，外12 SOD3。高度敏感的酶检测，包括这种检测SOD，蛋白质的交叉污染的可能性是可能的，需要进一步研究。误差棒代表教统局局长。

<ol>
	<li>Bishop, P. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+macromolecules+and+supramolecular+organisation+of+the+vitreous+gel.">Structural macromolecules and supramolecular organisation of the vitreous gel.</a> Prog. Retin. Eye Res. 19, 323-344 (2000).</li><li>Gao, B. B., Chen, X., Timothy, N., Aiello, L. P. &. a. m. p. ;. a. m. p., Feener, E. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+vitreous+proteome+in+diabetes+without+diabetic+retinopathy+and+diabetes+with+proliferative+diabetic+retinopathy.">Characterization of the vitreous proteome in diabetes without diabetic retinopathy and diabetes with proliferative diabetic retinopathy.</a> J. Proteome Res. 7, 2516-2525 (2008).</li><li>Izuta, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+SOD+and+VEGF+are+increased+in+vitreous+bodies+from+proliferative+diabetic+retinopathy+patients.">Extracellular SOD and VEGF are increased in vitreous bodies from proliferative diabetic retinopathy patients.</a> Mol. Vis. 15, 2663-2672 (2009).</li><li>Sebag, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+biology+of+pharmacologic+vitreolysis.">Molecular biology of pharmacologic vitreolysis.</a> Trans. Am. Ophthalmol. Soc. 103, 473-494 (2005).</li><li>Smith, R. S., Smith, R. S., John, S. W. M., Nishina, P. M., Sundberg, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Systematic evaluation of the mouse eye: Anatomy, pathology, and biomethods. , 161-195 (2002).</li><li>Ihanamaki, T., Metsaranta, M., Rintala, M., Vuorio, E., Sandberg-Lall, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ocular+abnormalities+in+transgenic+mice+harboring+mutations+in+the+type+II+collagen+gene.">Ocular abnormalities in transgenic mice harboring mutations in the type II collagen gene.</a> Eur. J. Ophthalmol. 6, 427-435 (1996).</li><li>Kaarniranta, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mouse+model+for+Stickler's+syndrome:+ocular+phenotype+of+mice+carrying+a+targeted+heterozygous+inactivation+of+type+II+(pro)collagen+gene+(Col2a1.">A mouse model for Stickler's syndrome: ocular phenotype of mice carrying a targeted heterozygous inactivation of type II (pro)collagen gene (Col2a1.</a> Exp. Eye Res. 83, 297-303 (2006).</li><li>Marneros, A. G. &. a. m. p. ;. a. m. p., Olsen, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-dependent+iris+abnormalities+in+collagen+XVIII/endostatin+deficient+mice+with+similarities+to+human+pigment+dispersion+syndrome.">Age-dependent iris abnormalities in collagen XVIII/endostatin deficient mice with similarities to human pigment dispersion syndrome.</a> Invest. Ophthalmol. Vis. Sci. 44, 2367-2372 (2003).</li><li>Martin, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogenesis+of+persistent+hyperplastic+primary+vitreous+in+mice+lacking+the+arf+tumor+suppressor+gene.">Pathogenesis of persistent hyperplastic primary vitreous in mice lacking the arf tumor suppressor gene.</a> Invest. Ophthalmol. Vis. Sci. 45, 3387-3396 (2004).</li><li>Song, B. J., Tsang, S. H., Lin, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+models+of+retinal+degeneration+and+targets+for+gene+therapy.">Genetic models of retinal degeneration and targets for gene therapy.</a> Gene. Ther. Mol. Biol. 11B, 229-262 (2007).</li><li>Skeie, J. M., Mahajan, V. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissection+of+Human+Vitreous+Body+Elements+for+Proteomic+Analysis.">Dissection of Human Vitreous Body Elements for Proteomic Analysis.</a> J Vis Exp. , (2011).</li><li>Ciechanowski, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impaired+synthesis+is+not+the+reason+for+decreased+activity+of+extracellular+superoxide+dismutase+in+patients+with+diabetes.">Impaired synthesis is not the reason for decreased activity of extracellular superoxide dismutase in patients with diabetes.</a> Arch Med Res. 36, 148-153 (2005).</li></ol>

按照协会的研究和视觉眼科眼科及视觉研究中使用动物的声明进行了动物实验。

然而，转基因小鼠视网膜和玻璃体视网膜疾病调查的重要模式。6-10鼠标玻璃体，包括比较由于在小鼠眼大镜头玻璃体人类眼睛的比例明显较小。5这使得很难分离和纯化鼠标玻璃体。了解蛋白质与疾病，如增生性糖尿病视网膜病变，年龄相关性黄斑变性，葡萄膜炎，视网膜脱离玻璃体变化会给机制的洞察力，他们的进步。这种可视化的实验协议，提供了一种手段获取和净化鼠标玻璃体，同时最大限度的酶和蛋白组学分析的蛋白质产量。

<table border="1"><tbody><tr><th>名称</th><th>公司</th><th>产品编号</th><th>评论</th></tr><tr><td> 15 °，BD海狸显微刀片</td><td>流式细胞 - 迪金森</td><td> 374881 </td><td></td></tr><tr><td> PBS，pH值7.4 </td><td> Invitrogen公司</td><td> 70011-044 </td><td></td></tr><tr><td> 0.22 Castroviejo精细缝合钳</td><td> STORZ Ophthalmics </td><td> E1805 </td><td></td></tr><tr><td> 0.12科利柏钳</td><td> STORZ Ophthalmics </td><td> 2 / 132 </td><td></td></tr><tr><td>单片机离心过滤Ultracel YM - 100或Y​​M - 50 </td><td> Millipore公司</td><td> 42412，42415 </td><td></td></tr><tr><td> SOD活性比色法检测试剂盒</td><td> Abcam公司</td><td> Ab65354 </td><td></td></tr><tr><td> Weck - CEL手术矛</td><td>美敦力公司</td><td> 0008680 </td><td></td></tr><tr><td>蛋白酶抑制剂的鸡尾酒</td><td>罗氏公司</td><td> 11 836 170 001 </td><td></td></tr><tr><td>火烈鸟荧光凝胶染色</td><td> Bio - Rad公司</td><td> 161-04910 </td><td></td></tr></tbody></table>

evisceration of mouse vitreous and retina for proteomic analyses

虽然作为一个重要的遗传性视网膜疾病的遗传模型小鼠视网膜出现，鼠标玻璃体仍然有待探索。玻璃体是一个高水覆人工以及眼蛋白质在疾病过程中积累的视网膜细胞外基质 。1-3异常玻璃体和视网膜之间的相互作用的几种疾病，如视网膜脱离，增殖性糖尿病视网膜病变，葡萄膜炎，和增生性玻璃体视网膜病变的基础。 4相对鼠标玻璃体体积明显比人类的玻璃体（图1），是因为鼠标镜头中占有近75％的眼睛。5这使得鼠标玻璃体生化研究具有挑战性。在这个视频文章中，我们目前的技术来剖析和隔离鼠标玻璃体视网膜，这将允许使用转基因小鼠模型，以更清楚地界定这种细胞外基质的作用，在玻璃体视网膜疾病的发展。

Watch this Scientific Journal Video about Evisceration of Mouse Vitreous and Retina for Proteomic Analyses at JoVE.com

Evisceration of Mouse Vitreous and Retina for Proteomic Analyses

剥离技术说明玻璃体，视网膜，并从小鼠眼晶状体，通过离心分离，并与蛋白质分析表征剜。

眼内容剜除小鼠玻璃体和视网膜蛋白质组分析

While the mouse retina has emerged as an important genetic model for inherited retinal disease, the mouse vitreous remains to be explored. The vitreous is ...

evisceration-of-mouse-vitreous-and-retina-for-proteomic-analyses

Research

JoVE Journal

Biology

28.9K 次观看.  University of Iowa. 剥离技术说明玻璃体，视网膜，并从小鼠眼晶状体，通过离心分离，并与蛋白质分析表征剜。

视频: Evisceration of Mouse Vitreous and Retina for Proteomic Analyses

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

Immunoblot Analysis

Immunoblotting (western blotting) is a rapid and sensitive assay for the detection and characterization of proteins that works by exploiting the specificity inherent in antigen-antibody recognition. It involves the solubilization and electrophoretic separation of proteins, glycoproteins, or lipopolysaccharides by gel electrophoresis, followed by quantitative transfer and irreversible binding to nitrocellulose, PVDF, or nylon. The immunoblotting technique has been useful in identifying specific antigens recognized by polyclonal or monoclonal antibodies and is highly sensitive (1 ng of antigen can be detected). This unit provides protocols for protein separation, blotting proteins onto membranes, immunoprobing, and visualization using chromogenic or chemiluminescent substrates.

Immunoblotting (western blotting) is a rapid and sensitive assay for the detection and characterization of proteins that works by exploiting the specificity inherent in antigen-antibody recognition. This video provides protocols for protein separation, blotting proteins onto membranes, immunoprobing, and visualization using chromogenic or chemiluminescent substrates.

免疫印迹分析

Immunoblotting (western blotting) is a rapid and sensitive assay for the detection and characterization of proteins that works by exploiting the ...

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 ...

Digital Microfluidics for Automated Proteomic Processing

Clinical proteomics has emerged as an important new discipline, promising the discovery of biomarkers that will be useful for early diagnosis and prognosis of disease. While clinical proteomic methods vary widely, a common characteristic is the need for (i) extraction of proteins from extremely heterogeneous fluids (i.e. serum, whole blood, etc.) and (ii) extensive biochemical processing prior to analysis. Here, we report a new digital microfluidics (DMF) based method integrating several processing steps used in clinical proteomics. This includes protein extraction, resolubilization, reduction, alkylation and enzymatic digestion. Digital microfluidics is a microscale fluid-handling technique in which nanoliter-microliter sized droplets are manipulated on an open surface. Droplets are positioned on top of an array of electrodes that are coated by a dielectric layer - when an electrical potential is applied to the droplet, charges accumulate on either side of the dielectric. The charges serve as electrostatic handles that can be used to control droplet position, and by biasing a sequence of electrodes in series, droplets can be made to dispense, move, merge, mix, and split on the surface. Therefore, DMF is a natural fit for carrying rapid, sequential, multistep, miniaturized automated biochemical assays. This represents a significant advance over conventional methods (relying on manual pipetting or robots), and has the potential to be a useful new tool in clinical proteomics.
Mais J. Jebrail, Vivienne N. Luk, and Steve C. C. Shih contributed equally to this work.
Sergio L. S. Freire's current address is at the University of the Sciences in Philadelphia located at 600 South 43rd Street, Philadelphia, PA 19104.

Digital Microfluidics is a technique characterized by the manipulation of discrete droplets (~nL - mL) on an array of electrodes by the application of electrical fields. It is well-suited for carrying out rapid, sequential, miniaturized automated biochemical assays. Here, we report a platform capable of automating several proteomic processing steps.

数字微流体进行自动蛋白质组学处理

Clinical proteomics has emerged as an important new discipline, promising the discovery of biomarkers that will be useful for early diagnosis and ...

Toxin Induction and Protein Extraction from Fusarium spp. Cultures for Proteomic Studies

Fusaria are filamentous fungi able to produce different toxins. Fusarium mycotoxins such as deoxynivalenol, nivalenol, T2, zearelenone, fusaric acid, moniliformin, etc... have adverse effects on both human and animal health and some are considered as pathogenicity factors. Proteomic studies showed to be effective for deciphering toxin production mechanisms (Taylor et al., 2008) as well as for identifying potential pathogenic factors (Paper et al., 2007, Houterman et al., 2007) in Fusaria. It becomes therefore fundamental to establish reliable methods for comparing between proteomic studies in order to rely on true differences found in protein expression among experiments, strains and laboratories. The procedure that will be described should contribute to an increased level of standardization of proteomic procedures by two ways. The filmed protocol is used to increase the level of details that can be described precisely. Moreover, the availability of standardized procedures to process biological replicates should guarantee a higher robustness of data, taking into account also the human factor within the technical reproducibility of the extraction procedure.
The protocol described requires 16 days for its completion: fourteen days for cultures and two days for protein extraction (figure 1). 
Briefly, Fusarium strains are grown on solid media for 4 days; they are then manually fragmented and transferred into a modified toxin inducing media (Jiao et al., 2008) for 10 days. Mycelium is collected by filtration through a Miracloth layer. Grinding is performed in a cold chamber. Different operators performed extraction replicates (n=3) in order to take into account the bias due to technical variations (figure 2). Extraction was based on a SDS/DTT buffer as described in Taylor et al. (2008) with slight modifications. Total protein extraction required a precipitation process of the proteins using Aceton/TCA/DTT buffer overnight and Acetone /DTT washing (figure 3a,3b). Proteins were finally resolubilized in the protein-labelling buffer and quantified. Results of the extraction were visualized on a 1D gel (Figure 4, SDS-PAGE), before proceeding to 2D gels (IEF/SDS-PAGE). The same procedure can be applied for proteomic analyses on other growing media and other filamentous fungi (Miles et al., 2007).

Toxin Induction and Protein Extraction from Fusarium spp. Cultures for Proteomic Studies

Protein extraction for proteomic analyses in fungal species requires high levels of standardization to be accomplished according with the minimum information about a proteomic experiment (MIAPE) guidelines. We present a video-protocol that includes a procedure for minimizing experimental bias during toxin induction and protein extraction from Fusarium spp.

毒素诱导和蛋白质提取镰刀 SPP。</em蛋白质组学研究>文化

Fusaria are filamentous fungi able to produce different toxins. Fusarium mycotoxins such as deoxynivalenol, nivalenol, T2, zearelenone, fusaric acid, ...

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Human cancer and response to therapy is better represented in orthotopic animal models. This paper describes the development of an orthotopic mouse model of ovarian cancer, treatment of cancer via oral delivery of drugs, and monitoring of tumor cell behavior in response to drug treatment in real time using in vivo imaging system. In this orthotopic model, ovarian tumor cells expressing luciferase are applied topically by injecting them directly into the mouse bursa where each ovary is enclosed. Upon injection of D-luciferin, a substrate of firefly luciferase, luciferase-expressing cells generate bioluminescence signals. This signal is detected by the in vivo imaging system and allows for a non-invasive means of monitoring tumor growth, distribution, and regression in individual animals. Drug administration via oral gavage allows for a maximum dosing volume of 10 mL/kg body weight to be delivered directly to the stomach and closely resembles delivery of drugs in clinical treatments. Therefore, techniques described here, development of an orthotopic mouse model of ovarian cancer, oral delivery of drugs, and in vivo imaging, are useful for better understanding of human ovarian cancer and treatment and will improve targeting this disease.

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy. A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells. Treatment is administered via oral gavage. Tumor growth is monitored by in vivo imaging system.

活体成像和治疗卵巢癌的原位小鼠模型

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

Isolation of Translating Ribosomes Containing Peptidyl-tRNAs for Functional and Structural Analyses

Recently, structural and biochemical studies have detailed many of the molecular events that occur in the ribosome during inhibition of protein synthesis by antibiotics and during nascent polypeptide synthesis. Some of these antibiotics, and regulatory nascent polypeptides mostly in the form of peptidyl-tRNAs, inhibit either peptide bond formation or translation termination1-7. These inhibitory events can stop the movement of the ribosome, a phenomenon termed "translational arrest". Translation arrest induced by either an antibiotic or a nascent polypeptide has been shown to regulate the expression of genes involved in diverse cellular functions such as cell growth, antibiotic resistance, protein translocation and cell metabolism8-13. Knowledge of how antibiotics and regulatory nascent polypeptides alter ribosome function is essential if we are to understand the complete role of the ribosome in translation, in every organism.
Here, we describe a simple methodology that can be used to purify, exclusively, for analysis, those ribosomes translating a specific mRNA and containing a specific peptidyl-tRNA14. This procedure is based on selective isolation of translating ribosomes bound to a biotin-labeled mRNA. These translational complexes are separated from other ribosomes in the same mixture, using streptavidin paramagnetic beads (SMB) and a magnetic field (MF). Biotin-labeled mRNAs are synthesized by run-off transcription assays using as templates PCR-generated DNA fragments that contain T7 transcriptional promoters. T7 RNA polymerase incorporates biotin-16-UMP from biotin-UTP; under our conditions approximately ten biotin-16-UMP molecules are incorporated in a 600 nt mRNA with a 25% UMP content. These biotin-labeled mRNAs are then isolated, and used in in vitro translation assays performed with release factor 2 (RF2)-depleted cell-free extracts obtained from Escherichia coli strains containing wild type or mutant ribosomes. Ribosomes translating the biotin-labeled mRNA sequences are stalled at the stop codon region, due to the absence of the RF2 protein, which normally accomplishes translation termination. Stalled ribosomes containing the newly synthesized peptidyl-tRNA are isolated and removed from the translation reactions using SMB and an MF. These beads only bind biotin-containing messages.
The isolated, translational complexes, can be used to analyze the structural and functional features of wild type or mutant ribosomal components, or peptidyl-tRNA sequences, as well as determining ribosome interaction with antibiotics or other molecular factors 1,14-16. To examine the function of these isolated ribosome complexes, peptidyl-transferase assays can be performed in the presence of the antibiotic puromycin1. To study structural changes in translational complexes, well established procedures can be used, such as i) crosslinking to specific amino acids14 and/or ii) alkylation protection assays1,14,17.

A major impediment to biochemical analyses of ribosomes containing nascent peptidyl-tRNAs has been the presence of other ribosomes in the same samples, ribosomes not involved in the translation of the specific mRNA sequence being analyzed. We developed a simple methodology to purify, exclusively, the ribosomes containing the nascent peptidyl-tRNA of interest.

分离翻译含肽- tRNA的核糖体的功能和结构分析

Recently, structural and biochemical studies have detailed many of the molecular events that occur in the ribosome during inhibition of protein synthesis ...

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal neurons. In contrast, the adult zebrafish retina possesses the robust ability to spontaneously regenerate any neuronal class that is lost in a variety of different retinal damage models, including retinal puncture, chemical ablation, concentrated high temperature, and intense light treatment 1-8. Our lab extensively characterized regeneration of photoreceptors following constant intense light treatment and inner retinal neurons after intravitreal ouabain injection 2, 5, 9. In all cases, resident M&uuml;ller glia re-enter the cell cycle to produce neuronal progenitors, which continue to proliferate and migrate to the proper retinal layer, where they differentiate into the deficient neurons. 
	We characterized five different stages during regeneration of the light-damaged retina that were highlighted by specific cellular responses. We identified several differentially expressed genes at each stage of retinal regeneration by mRNA microarray analysis 10. Many of these genes are also critical for ocular development. To test the role of each candidate gene/protein during retinal regeneration, we needed to develop a method to conditionally limit the expression of a candidate protein only at times during regeneration of the adult retina. 
	Morpholino oligos are widely used to study loss of function of specific proteins during the development of zebrafish, Xenopus, chick, mouse, and tumors in human xenografts 11-14. These modified oligos basepair with complementary RNA sequence to either block the splicing or translation of the target RNA. Morpholinos are stable in the cell and can eliminate or "knockdown" protein expression for three to five days 12. 
	Here, we describe a method to efficiently knockdown target protein expression in the adult zebrafish retina. This method employs lissamine-tagged antisense morpholinos that are injected into the vitreous of the adult zebrafish eye. Using electrode forceps, the morpholino is then electroporated into all the cell types of the dorsal and central retina. Lissamine provides the charge on the morpholino for electroporation and can be visualized to assess the presence of the morpholino in the retinal cells. 
	Conditional knockdown in the retina can be used to examine the role of specific proteins at different times during regeneration. Additionally, this approach can be used to study the role of specific proteins in the undamaged retina, in such processes as visual transduction and visual processing in second order neurons.

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

A method to conditionally knockdown a target protein&#x2019;s expression in the adult zebrafish retina is described, which involves intravitreally injecting antisense morpholinos and electroporating them into the retina. The resulting protein is knocked down for several days, which allows testing the protein&#x2019;s role in the regenerating or intact retina.

在体内电穿孔Morpholinos到成年斑马鱼视网膜

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal ...

Trajectory Data Analyses for Pedestrian Space-time Activity Study

It is well recognized that human movement in the spatial and temporal dimensions has direct influence on disease transmission1-3. An infectious disease typically spreads via contact between infected and susceptible individuals in their overlapped activity spaces. Therefore, daily mobility-activity information can be used as an indicator to measure exposures to risk factors of infection. However, a major difficulty and thus the reason for paucity of studies of infectious disease transmission at the micro scale arise from the lack of detailed individual mobility data. Previously in transportation and tourism research detailed space-time activity data often relied on the time-space diary technique, which requires subjects to actively record their activities in time and space. This is highly demanding for the participants and collaboration from the participants greatly affects the quality of data4.
Modern technologies such as GPS and mobile communications have made possible the automatic collection of trajectory data. The data collected, however, is not ideal for modeling human space-time activities, limited by the accuracies of existing devices. There is also no readily available tool for efficient processing of the data for human behavior study. We present here a suite of methods and an integrated ArcGIS desktop-based visual interface for the pre-processing and spatiotemporal analyses of trajectory data. We provide examples of how such processing may be used to model human space-time activities, especially with error-rich pedestrian trajectory data, that could be useful in public health studies such as infectious disease transmission modeling.
The procedure presented includes pre-processing, trajectory segmentation, activity space characterization, density estimation and visualization, and a few other exploratory analysis methods. Pre-processing is the cleaning of noisy raw trajectory data. We introduce an interactive visual pre-processing interface as well as an automatic module. Trajectory segmentation5 involves the identification of indoor and outdoor parts from pre-processed space-time tracks. Again, both interactive visual segmentation and automatic segmentation are supported. Segmented space-time tracks are then analyzed to derive characteristics of one's activity space such as activity radius etc. Density estimation and visualization are used to examine large amount of trajectory data to model hot spots and interactions. We demonstrate both density surface mapping6 and density volume rendering7. We also include a couple of other exploratory data analyses (EDA) and visualizations tools, such as Google Earth animation support and connection analysis. The suite of analytical as well as visual methods presented in this paper may be applied to any trajectory data for space-time activity studies.

A suite of spatiotemporal processing methods are presented to analyze human trajectory data, such as that collected using a GPS device, for the purpose of modeling pedestrian space-time activities.

轨迹数据分析行人时空活性研究

It is well recognized that human movement in the  spatial and temporal dimensions has direct influence on disease transmission1-3.  An infectious disease ...

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling processes. Extracting sterol-enriched membrane microdomains from plant cells for proteomic analysis is a difficult task mainly due to multiple preparation steps and sources for contaminations from other cellular compartments. The plasma membrane constitutes only about 5-20% of all the membranes in a plant cell, and therefore isolation of highly purified plasma membrane fraction is challenging. A frequently used method involves aqueous two-phase partitioning in polyethylene glycol and dextran, which yields plasma membrane vesicles with a purity of 95% 1. Sterol-rich membrane microdomains within the plasma membrane are insoluble upon treatment with cold nonionic detergents at alkaline pH. This detergent-resistant membrane fraction can be separated from the bulk plasma membrane by ultracentrifugation in a sucrose gradient 2. Subsequently, proteins can be extracted from the low density band of the sucrose gradient by methanol/chloroform precipitation. Extracted protein will then be trypsin digested, desalted and finally analyzed by LC-MS/MS. Our extraction protocol for sterol-rich microdomains is optimized for the preparation of clean detergent-resistant membrane fractions from Arabidopsis thaliana cell cultures.
We use full metabolic labeling of Arabidopsis thaliana suspension cell cultures with K15NO3 as the only nitrogen source for quantitative comparative proteomic studies following biological treatment of interest 3. By mixing equal ratios of labeled and unlabeled cell cultures for joint protein extraction the influence of preparation steps on final quantitative result is kept at a minimum. Also loss of material during extraction will affect both control and treatment samples in the same way, and therefore the ratio of light and heave peptide will remain constant. In the proposed method either labeled or unlabeled cell culture undergoes a biological treatment, while the other serves as control 4.

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Here we describe a robust method for the fractionation of plant plasma membranes into detergent resistant and detergent soluble membranes based on a mixture of unlabeled and in vivo fully 15N labeled Arabidopsis thaliana cell cultures. The procedure is applied for comparative proteomic studies to understand signaling processes.

代谢标记和膜分馏的比较蛋白质组学分析<em&gt;拟南芥</em&gt;悬浮细胞培养

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling ...

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眼内容剜除小鼠玻璃体和视网膜蛋白质组分析