기금은 시력과 실명을 방지하기 위해 연구 전투에 의해 제공되었다.

1. 앞쪽에 세그먼트 해부. <ol><li> limbus에 후부 Scleral 조직은 0.22 forcep과 세계 (눈 공) 안정화는 함께 파악합니다. </li><li> microsurgical 블레이드는 limbus에서 limbus로 각막의 선형 절개를하는 데 사용됩니다. </li><li> 0.12 콜리 브리 forcep는 다음 절개에서 각막을 파악하는 데 사용됩니다. </li><li> 앞쪽에 챔버 유체는 다음 샌드위치 - 셀 수술 창으로 흡수됩니다. </li></ol> 2. 렌즈 내장 적출. <ol><li> 각막 쥐고 0.12 콜리 브리 forcep는 세계를 안정화하는 데 사용됩니다. </li><li> 멋진 곡선 바늘 홀더 (또는 곡선 드레싱 포셉) 지구의 후부 측면 방향으로 렌즈 뒤에 삽입됩니다. 바늘 홀더가 부분적으로 폐쇄하고, 앞으로 가져온 것입니다. 압력이 안구 벽은 그대로 둔 상태에서 각막 절개를 통해 앞으로 렌즈를 못살게 굴지 공막엔의 외부 표면에 포셉를 사용하여 적용됩니다. 유리체는 반투명 젤로 표시하고 렌즈에 부분적으로 자기편이다. </li><li> 렌즈 - 유리의 조직​​은 다음 PBS에 용해 테아제 억제제 칵테일 (로체) 20 microliters을 포함하는 필터링 원심 분리 튜브에 배치됩니다. </li></ol> 3. 망막의 내장 적출. <ol><li> 각막 절개를 쥐고 0.12 콜리 브리의 집게와 함께 지구를 안정화하기 위해 계속합니다. </li><li> 멋진 곡선 바늘 홀더 (또는 곡선 드레싱 포셉) 시신경 근처 가능한 세계에까지 후부로 배치됩니다. 바늘 홀더가 부분적으로 폐쇄하고, 앞으로 가져온 것입니다. 압력은 각막 절개를 통해 앞으로 망막들을 못살게 굴지 공막엔의 외부 표면에 포셉를 사용하여 적용됩니다. 유리체가 망막에 자기편 반투명 젤로 나타납니다. 망막은 노란색, vascularized 조직으로 시각이다. 색소 조직의 스트립은 집게로 멀리 벗겨 수 있습니다. </li><li> 망막 - 유리체 조직은 다음 렌즈 - 유리 조직을 포함하는 필터링 원심 튜브에 배치됩니다. </li></ol> 4. 원심 분리를 필터링. <ol><li> 여과 원심 분리기 튜브는 benchtop의 원심 분리기에 배치됩니다. 12분에 대한 14,000 X G에서 스핀. </li><li> 유리체있는 하부 챔버에서 eluant을 대기음. 망막은 상부 챔버에 남아 있습니다. 이 샘플은 단백질 연구를 위해 이용하실 수 있습니다. </li></ol> 5. 대표 결과 유리 샘플은 SDS - PAGE (그림 2)에 의해 분석되었다. 샘플은 또한 효소 활동 분석 (그림 3)에 사용되었습니다. <img src="/files/ftp_upload/2795/2795fig1.jpg" alt="그림 1"/> 그림 1. 마우스 눈 다이어그램. 인간과 마우스 눈 교차 단면 개략도는 큰 렌즈로 인해 마우스 눈을 상대 작은 유리 볼륨을 보여줍니다. <img src="/files/ftp_upload/2795/2795fig2.jpg" alt="그림 2"/> 그림 2. 한 차원 SDS - PAGE 마우스 유리체 및 망막의. 단백질 마우스 유리의 프로필, 13.6 MG / ML (레인 1), 그리고 망막, 11.35 MG / ML (레인 2). 젤 전기 영동은 플라밍고 형광 겔 얼룩 물들일, 45 분 150 KV에서 공연하고 VersaDoc 이미징 시스템 (BioRad, 헤라클레스, CA)를 사용하여 시각되었습니다. <img src="/files/ftp_upload/2795/2795fig3.jpg" alt="그림 3"/> 그림 3. 마우스와 인간의 유리체에서 초과 산화물의 dismutase (소드) 효소 활동. 총 소드 활동의 측정 (소드 활동 colorimetric aasay 키트, # AB65354, Abcam, 병원, 캠브리지, MA) 유리와 DBA와 흰둥이 생쥐에서 내장 적출에 의해 수집된 망막에서 수행되었다. 이것은 인간의 유리 코어 및 상대적인 활동을 결정하기 위해 사후 인간 기증자 눈을에서 수집한 인간의 망막에있는 소드 활동에 비해했다. Undiluted 유리체 코어 샘플은 수동으로 23 게이지 바늘, 11, 망막은 눈 꽃과 거리 RPE / 맥락막 복잡한에서 조직을 절단하여 수집되었습니다을 사용 aspirated했다. 분석은 소드 isoforms 구분하지는 않지만 그것이 유일한 세포 이소형이기 때문에, 유리체 소드 활동, SOD3을 반영 가능성이 12 망막 소드 활동이 세 소드 isoforms을 반영 가능성이 있습니다. 세포내 SOD1, mitochondrial SOD2, 그리고 세포 SOD3가. 12이 소드 분석을 포함하여 고도로 민감한 효소 assays, 들어, 단백질의 교차 오염의 가능성이 가능하고 추가 연구를 필요로합니다. 오류 막대는 SEM을 나타냅니다.

<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. 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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>

동물 실험은 안과 및 비전 연구에 동물의 사용에 대한 안과 정책에 연구 및 비전 협회에 따라 수행되었다.

유전자 변형 생쥐는 망막과 vitreoretinal 질병을 조사하는 중요한 모델입니다. 6-10는 마우스 유리 본체 그러나, 마우스 눈 속에있는 대형 렌즈로 인한 인간의 유리에 비해 눈 크게 작은 비율로 구성되어 있습니다. 5이 있습니다 그것은 어려운 마우스 유리를 분리하고 정화합니다. 이러한 proliferative 당뇨성 망막 병증의, 나이 관련 황반 변성, uveitis, 그리고 망막 분리와 같은 질병 중 유리체와 관련된 단백질의 변화를 이해하는 것은 메커니즘에 대한 통찰력을 제공합니다 어떤 사람들이 진행하여. 효소 및 proteomic 분석을위한 단백질 수율을 극대화하면서이 시각 실험 프로토콜, 마우스 유리기구를 구하고 정화하기위한 수단을 제공합니다.

<table border="1"><tbody><tr><th> 이름 </th><th> 회사 </th><th> 카탈로그 번호 </th><th> 댓글 </th></tr><tr><td> 15 °, BD 비버 Microsurgical 블레이드 </td><td> 백톤 디킨슨 - </td><td> 374881 </td><td></td></tr><tr><td> PBS, 산도 7.4 </td><td> Invitrogen </td><td> 70011-044 </td><td></td></tr><tr><td> 0.22 미세 Castroviejo Suturing의 포셉 </td><td> Storz Ophthalmics </td><td> E1805 </td><td></td></tr><tr><td> 0.12 콜리 브리 포셉 </td><td> Storz Ophthalmics </td><td> 132분의 2 </td><td></td></tr><tr><td> Microcon 원심 필터 Ultracel YM - 100 YM - 50 </td><td> Millipore </td><td> 42412, 42415 </td><td></td></tr><tr><td> 소드 활동 Colorimetric 분석 키트 </td><td> Abcam </td><td> Ab65354 </td><td></td></tr><tr><td> 샌드위치 - 셀 외과 스피어스 </td><td> 메드 트로닉 </td><td> 0,008,680 </td><td></td></tr><tr><td> 프로 테아제 억제제 칵테일 </td><td> 로체 </td><td> 11 836 170 001 </td><td></td></tr><tr><td> 플라밍고 형광 젤은 얼룩 </td><td> 바이오 래드 </td><td> 161-04910 </td><td></td></tr></tbody></table>

evisceration of mouse vitreous and retina for proteomic analyses

마우스 망막은 상속 망막 질환의 중요한 유전 모델로 등장했습니다 동안 마우스 유리는 탐험으로 남아 있습니다. 유리체는 intraocular뿐만 아니라 extraocular 단백질이 질병 중에 축적 망막을 overlying 매우 수성 세포외 기질이다. 유리체와 망막 사이의 1-3 비정상적인 상호 작용 이러한 망막 분리, proliferative 당뇨병의 망막 병증, uveitis, 그리고 proliferative vitreoretinopathy과 같은 여러 가지 질병을 기초 1. , 마우스 렌즈는 안구의 약 75 % 점유 이후 4 상대 마우스 유리 볼륨은 인간의 유리 (그림 1)보다 훨씬 작습니다. 5이 어려운 마우스 유리의 생화 학적 연구를 하였다. 이 동영상이 문서에서 우리는 유전자 변형 마우스 모델의 사용이 더 명확하게 vitreoretinal 질병의 개발이 세포외 기질의 역할을 정의할 수 망막에서 마우스 유리를 해부하다하고 분리하는 기술을 제시한다.

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

해부 기술은 유리체, 망막, 그리고 마우스 눈에서 렌즈, 원심 분리에 의해 분리하고, 단백질 as​​says과 특성화의 내장 적출을 보여줍니다.

Proteomic 분석을위한 마우스 유리체 및 망막의 내장 적출

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 조회수.  University of Iowa. 해부 기술은 유리체, 망막, 그리고 마우스 눈에서 렌즈, 원심 분리에 의해 분리하고, 단백질 as​​says과 특성화의 내장 적출을 보여줍니다.

비디오: 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).

단백질의 전기 영동 분리

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

연구

생물학

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.

Immunoblot 분석

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

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.

자동 Proteomic 처리를위한 디지털 Microfluidics

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.

에서 독소 유도와 단백질 추출 Fusarium 종.</emProteomic 연구> 문화

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.

생체내 이미징 및 난소암의 Orthotopic 마우스 모델에서 치료 트리 트먼트

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.

기능 및 구조 분석을위한 Peptidyl - tRNAs를 포함 번역 리보솜의 분리

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.

Morpholinos의 생체내의 Electroporation에서 성인 Zebrafish의 망막에

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.

보행자 공간 시간 활동 학습을위한 궤적 데이터 분석

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.

비교 단백체 분석을위한 신진 대사 라벨링 및 막 분별<em&gt; 애기 장대</em&gt; 서스펜션 세포 배양

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

Extraction of Venom and Venom Gland Microdissections from Spiders for Proteomic and Transcriptomic Analyses

Venoms are chemically complex secretions typically comprising numerous proteins and peptides with varied physiological activities. Functional characterization of venom proteins has important biomedical applications, including the identification of drug leads or probes for cellular receptors. Spiders are the most species rich clade of venomous organisms, but the venoms of only a few species are well-understood, in part due to the difficulty associated with collecting minute quantities of venom from small animals. This paper presents a protocol for the collection of venom from spiders using electrical stimulation, demonstrating the procedure on the Western black widow (Latrodectus hesperus). The collected venom is useful for varied downstream analyses including direct protein identification via mass spectrometry, functional assays, and stimulation of venom gene expression for transcriptomic studies. This technique has the advantage over protocols that isolate venom from whole gland homogenates, which do not separate genuine venom components from cellular proteins that are not secreted as part of the venom. Representative results demonstrate the detection of known venom peptides from the collected sample using mass spectrometry. The venom collection procedure is followed by a protocol for dissecting spider venom glands, with results demonstrating that this leads to the characterization of venom-expressed proteins and peptides at the sequence level.

This article provides a protocol for the extraction of venom from spiders using electrical stimulation in order to 1) conduct proteomic characterization, 2) stimulate venom gland gene expression, and 3) perform functional studies of venoms. This is followed by a description of venom gland microdissections for gene expression studies.