저자는 AH에 생명 수상 Un​​iversity of Florida의 과학 하워드 휴즈 의학 연구소 감사

1. 카세트를 준비 <ol><li> 클린 앞면과 뒷면 사용 Invitrogen Nupage Novex minigel 카세트, 또는 기타 상업적 minigel 카세트의 접시. </li><li> 플라스틱 에폭시의 약 1-3 ML을 준비하고 제조 업체의 지시에 따라 잘 섞는다. </li><li> 카세트가 조립되면 두 접시가 접촉되는 전면 판의 안쪽 가장자리에 에폭시를 적용합니다. </li><li> 오프닝을 막기에서 초과 에폭시을 방지하기 위해 카세트를 조립하면 다시 접시의 바닥 슬릿을 통해 필터 종이 또는 판지의 조각을 밀어 넣습니다. 즉시 두 접시가 조립되는대로 필터 종이를 제거합니다. </li><li> 꽉 도장을위한 가장자리를 따라 바인더 클립을 놓고 카세트가 완전히 치료 에폭시를 위해 하룻밤 사이에 앉자. </li><li> 전기 테이프로 다시 접시의 바닥에 슬릿을 봉쇄해. </li></ol> 2. 해결 젤을하고 있는걸 <ol><li> 125 ML 사이드 암 플라스크에 다음을 추가합니다4X 트리스 - 자료 해결 젤 버퍼 산도 8.70, 30.8 %의 아크릴 아미드 / bisacrylamide의 3.50 ML, 유형 3.25 ML의 2.25 ML 9 ML의 총 부피를 위해서 시약 학년 물 (IRG-H 2 O). </li><li> 드 가스 진공 장치로 플라스크의 측면 팔을 부착하고, 플라스크의 상단에 거꾸로 마개를 삽입하여 10 분, 대한​​ 솔루션입니다. </li><li> 50 ML 일회용 폴리 프로필렌의 원심 관에 드 기름 솔루션의 9​​ ML 전송 30 10 % ammounium의 persulfate (APS)의 μl와 N의 μl 6, N, N &#39;, N&#39;- tetramethylethylenediamine (TEMED)와 믹스를 추가 거품을 도입 피하기 위해 솔루션을 소용돌이에 의해 잘. </li><li> 바로 그것이 겔 접시 사이에 형성에서 모든 거품을 방지하기 위해 카세트의 측면을 실행할 수 있도록 카세트에 솔루션을 붓는다. 앞면 (짧은) 접시의 맨 가장자리에서 약 2cm로 카세트를 채우십시오. </li><li> 부드럽게 1X 해결 겔 버퍼로 포화 1-butanol 0.5 ML을 추가하고 젤은 1 중합하자시간. </li></ol> 3. 스태킹 젤을하고 있는걸 <ol><li> 4X 트리스베이스 스태킹 겔 버퍼 산도 6.80, 30.8 %의 아크릴 아미드 / bisacrylamide의 0.40 ML, 3 ML 총 볼륨 IRG-H 2 O의 1.85 ML의 0.75 ML : 125 ML 사이드 암 플라스크에 다음을 추가합니다. </li><li> 드 가스 단계는 2 단계에 설명된대로 10 분, 대한​​ 솔루션입니다. </li><li> 스태킹 겔 솔루션 degassing되는 동안 해결 젤 상단에서 1X 해결 젤 버퍼로 포화 1-butanol을 붓는다. IRG-H 2 O를 함께 한 해결 젤의 상단을 씻어 물을 붓고하고 남은 액체를 흡수하기 위해 물을 빨아들이는이나 워트 먼지 필터 용지를 사용합니다. </li><li> 당신의 degassed 솔루션 3 ML을 제거하고 10 %의 APS와 TEMED 2 μl 9 μl를 추가하고 잘 섞는다. </li><li> 신속하게 그것이 짧은 프런트 겔 판의 상단 가장자리 근처에까지 그것이 카세트의 측면을 실행할 수 있도록 카세트에 솔루션을 붓는다. </li><li> , 겔 카세트 상단으로 클린 겔 빗을를 삽입그 장소에서 개최 빗 위에 카세트에서 바인더 클립을 넣으십시오. 스태킹 겔은 하룻밤 사이에 1 시간 동안 중합하자. </li></ol> 4. 젤 저장 <ol><li> 겔 완전히 polymerized되면​​ 바인더 클립을 제거하고 열을 sealable 플라스틱 주머니에있는 카세트, 또는 다시 sealable 비닐 봉투를 넣습니다. </li><li> 주머니에 0.1 %의 나트륨 azide를 포함 1X 스태킹 겔 버퍼 20 ML 하거라. </li><li> 5 초 동안 주머니를 밀봉하고 봉투에는 누수가 없는지 확인 열정. </li><li> 4 ° C.에서 젤을 저장 </li></ol> 5. 샘플 준비 <ol><li> 얇은 벽으로 둘러싸인 microcentrifuge 튜브에서 단백질 샘플의 6.5 μl, 대한 2X Novex 트리스 - 글리신 SDS 샘플 버퍼, 100 MM dithiothreitol (DTT)과 IRG-H 2 O 1 μl (필요한 경우) 7.5 μl에 추가 15 μl의 총 부피. </li><li> vortexing하여 샘플을 섞어서하고, 간략하게 튜브의 바닥에 내용을 가져 샘플을 원심 분리기. </li> &lt;리&gt; 변성 70 샘플 ° C ~ 10 분입니다. </li></ol> 6. 젤 실행하기 <ol><li> Novex 겔 장치를 설정합니다. </li><li> 내부 챔버에 Nupage 항산화 500 μl, 그리고 트리스 - 글리신은 바깥 실로 버퍼를 실행하면 300 ML을 포함 트리스 - 글리신 나오는 버퍼 200 ML을 추가합니다. </li><li> 겔로드 피펫 팁을 사용하는 우물에 변성 단백질 샘플 (15 μl)을로드합니다. </li><li> 60-70 분 200 V에서 젤을 실행합니다. </li></ol> 7. 젤 염색법 <ol><li> 겔 장치에서 카세트를 제거하고 겔 나이프 또는 빳빳한 퍼티 나이프를 사용하여 별개의 두 플라스틱 겔 접시를 떼어 내려고하면 에폭시 도장을 깰. 젤 찢어하지 않도록 돌보는 접시에서 젤을 조심스럽게 분리. </li><li> IRG-H와 젤 부드러운 선동 2 O를 세 번 씻어. </li><li> 부드러운 교반과 1 시간 Invitrogen SimplyBlue SafeStain와 젤 청바지. </li><li> 염색법 솔루션을 붓고IRG-H 2 O를 가진 젤 destain </li></ol> 8. 카세트 청소 <ol><li> 그들이 다시 재사 용할 수 있도록 카세트의 모든 에폭시 긁어서거나 껍질하는 금속 주걱을 사용합니다. </li><li> 순한 세제 용액으로 플라스틱 겔 접시 각각 씻어하고, 증류수 다음 수돗물로 잘 씻어. </li></ol> 9. 대표 결과 에폭시가 물 방지 시일 (그림 1)을 형성하므로, 카세트는 아크릴 아미드 솔루션은 그들에 부어 때 누출되지 않습니다. 또한, 겔 카세트 쉽게 염색법을위한 젤을 검색할 열 수 있으며 에폭시들은 지속적인 재사용을 허용하기 위해 카세트 놔 벗겨 수 있습니다. 그림 2에 표시된 바와 같이, 젤은 단백질 마커와 일상 단백질 전기 영동에 적합한 샘플 밴드의 명확한 분리를 보여줍니다. <img src="/files/ftp_upload/3465/3465fig1.jpg" alt="그림 1" /> 1 그림. Appliminigel 카세트를 봉인하기 위해 에폭시의 양이온이. 에폭시의 박막 층을 조립 이전 카세트 중 하나의 가장자리에 적용됩니다. 에폭시를 강조하기 위해서는 해당 이미지의 허위 - 붉은 색이었다. <img src="/files/ftp_upload/3465/3465fig2.jpg" alt="그림 2" /> 그림 2. SDS-트리스 - 글리신 젤의 단백질의 분리는 다시 사용 minigel 카세트를 사용하여 부어. 위에서 설명한대로 SDS-PAGE이 수행되었다. 다음은 전기 영동은 겔이 제조 업체의 지침에 따라 SimplyBlue SafeStain으로 물들일 minigel 카세트, 제거 및 전송 하얀 빛을 이용하여 촬영되었다. 레인 M은 SeeBlue Plus2 사전 묻은 단백질 표준을 포함​​하고 있습니다. 차선 M의 단백질의 분자 무게는 kilodaltons (kDa)에서 젤 오른쪽에 표시됩니다. 다른 차선은 글루타티온-S-transferase (GST) - 태그가 단백질을 표현 박테리아로부터 허가 lysate에서 단백질을 보여줍니다. 표시된 단백질은 g에서 씻어 복이 있나니GST-태그를 단백질의 용출 이전 lutathione 비즈.

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

관심의 어떠한 충돌 선언 없습니다.

단백질 겔 전기 영동은 대부분의 분자 생물학 연구소를위한 indispensible 방법입니다. 상용 minigels의 편의를 높은 수준을 제공하지만, 치명타가 될 수 있습니다. 여기 상용 minigels의 인기 브랜드에서 minigel 카세트를 다시 사용하는 방법을 보여줍니다. 이 방법은 젤의 기성품 소스를 가지고있는 편리함을 유지하지만, 상용 minigels 비용의 분수에. 이 절차의 주요 단계는 완전히 유출을 방지하기 위해 unpo...

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 댓글 </td></tr><tr><td> Invitrogen Nupage Novex Minigel 카세트 (1.0 ㎜) </td><td> Invitrogen </td><td> NC2010 </td><td> 2, 5, 10, 12, 15 및 2D 우물에서 사용 가능 </td></tr><tr><td> 플라스틱 에폭시 </td><td> Loctite </td><td> 108,771 </td><td></td></tr><tr><td> 전기 테이프 </td><td> 스카치 </td><td></td><td></td></tr><tr><td> 125 ML 사이드 암 플라스크 </td><td> Pyrex </td><td> 5360 </td><td></td></tr><tr><td> 4X 트리스베이스 해결 겔 버퍼 </td><td></td><td></td><td> 1.5 M 트리스 -베이스 산도 8.7 </td></tr><tr><td> 아크릴 아미드 </td><td> 피셔 과학 </td><td> 01065-500 </td><td></td></tr><tr><td> 비스 아크릴 아미드 </td><td> MP의 Biomedicals 주식 회사 </td><td> 800,173 </td><td></td></tr><tr><td> 30.8 %의 아크릴 아미드 용액 </td><td></td><td></td><td> 30:0.8 아크릴 아미드 : 비스 아크릴 아미드 IRG-H 2 O의 </td></tr><tr><td> 10 % APS </td><td> 피셔 과학 </td><td> BP179-25 </td><td> 한 ML IRG-H 2 O에 0.1 g의 암모늄 persulfate는 사용하기 전에 즉시 준비 </td></tr><tr><td> TEMED </td><td> Arcos의 생명체 </td><td> 138,450,500 </td><td></td></tr><tr><td> 1-butanol 1X 해결 젤 버퍼로 포화 </td><td></td><td></td><td> 1X 해결 겔 버퍼에 5시 2분는 1-butanol </td></tr><tr><td> 4X 트리스베이스 스태킹 겔 버퍼 </td><td></td><td></td><td> 1.5 M 트리스 -베이스 산도 6.8 </td></tr><tr><td> 젤 빗 </td><td> Invitrogen </td><td> NC3015 </td><td> 15 음, 1.0 밀리미터 두께 </td></tr><tr><td> 충동 실러 </td><td> TEW 전기 난방 장비 </td><td></td><td></td></tr><tr><td> 히트 Sealable 파우치 </td><td> Kapak / Scotchpak </td><td></td><td> 1 파인트 크기 (6.5 &quot;X 8&quot;) </td></tr><tr><td> 젤 저장 버퍼 </td><td></td><td></td><td> 1X는 0.1 %의 나트륨 azide와 겔 버퍼를 스태킹 </td></tr><tr><td> Novex SDS 트리스 - 글리신 샘플 버퍼 (2X) </td><td> Invitrogen </td><td> LC2676 </td><td></td></tr><tr><td> DTT </td><td> FisherBiotech </td><td> BP172-5 </td><td></td></tr><tr><td> 10X 트리스 - Gylcine 나오는 버퍼 </td><td> Invitrogen </td><td> LC2675 </td><td> 10X 재고 1L : 29.0 g 트리스베이스, 144.0 g 글리신, 10.0 g SDS </td></tr><tr><td> XCell SureLock 미니 셀 </td><td> Invitrogen </td><td> EI0001 </td><td></td></tr><tr><td> 젤 나이프 </td><td> Invitrogen </td><td> EI9010 </td><td></td></tr><tr><td> SimplyBlue 금고 청바지 </td><td> Invitrogen </td><td> LC6065 </tD&gt; <td></td></tr></tbody></table>

pouring and running a protein gel by reusing commercial cassettes

나트륨 dodecyl 황산염의 polyacrylamide 겔 전기 영동 (SDS-PAGE) 분석을 사용하여 단백질의 평가는 생화학 및 분자 생물학 연구 1-4에서 사용하는 일반적인 기법입니다. 단백질의 일상 분석을 수행하는 실험실의 경우 상용 polyacrylamide의 젤 (~ $ 10/gel)의 비용은 시간의 경과에 상당한 될 수 있습니다. 이 비용을 완화하기 위해 일부 연구자들은 자신의 polyacrylamide 젤을 준비합니다. 이러한 젤류를 따르고 전통적인 방법은 일반적으로 전문 장비와 비용이 많은 젤류를 따르고과 향후 사용을 위해 그들을 저장할 수 있습니다 배제 유리 겔 접시를 사용합니다. 또한, 붓는 청소 또는 젤 동안 유리 조각으로 취급하는 것은 대학 연​​구실 교실에서 자신들의 사용을 배제 있습니다 안전 위험을 작성, 우발적인 파손이 발생할 수 있습니다. 우리의 프로토콜은 재활용 Invitrogen Nupage Novex minigel 카세트에 의해 동시에 여러 단백질 젤을 잡다, 그리고 저렴한 mA하는 방법을 보여줍니다terials는 인테리어 가게에서 구입했습니다. 이것은 경제적이고 능률적인 방법은 몇 주 동안 4 ° C에서 젤을 저장하는 방법을 포함하고 있습니다. 상용 젤, 자주 단백질 젤을 실행 실험실에서 플라스틱 겔 카세트를 다시 사용함으로써 상당한 비용을 절약하고 환경에 도움을 줄 수 있습니다. 또한, 플라스틱 겔 카세트는 학부 실험실 교실에 이상적하게 파손에 매우 저항력이 있습니다.

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

Pouring and Running a Protein Gel by reusing Commercial Cassettes

우리의 프로토콜은 홈 개선 스토어에서 구입 재활용 Invitrogen Nupage Novex minigel 카세트, 그리고 저렴한 재료로 한 번에 여러 개의 단백질 젤을 부어하는 방법을 보여줍니다. 이것은 경제적이고 능률적인 방법은 몇 주 동안 4 ° C에서 젤을 저장하는 방법을 포함하고 있습니다. 상용 젤, 자주 단백질 젤을 실행 실험실에서 플라스틱 겔 카세트를 다시 사용함으로써 상당한 비용을 절약하고 환경에 도움을 줄 수 있습니다.

상업 카세트를 재사용하여 물을 붓는과 단백질 젤 러닝

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

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JoVE Journal

Biology

24.8K 조회수.  University of Florida. 우리의 프로토콜은 홈 개선 스토어에서 구입 재활용 Invitrogen Nupage Novex minigel 카세트, 그리고 저렴한 재료로 한 번에 여러 개의 단백질 젤을 부어하는 방법을 보여줍니다. 이것은 경제적이고 능률적인 방법은 몇 주 동안 4 ° C에서 젤을 저장하는 방법을 포함하고 있습니다. 상용 젤, 자주 단백질 젤을 실행 실험실에서 플라스틱 겔 카세트를 다시 사용함으로써 ?...

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

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

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

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

세미 Denaturing 세제 - 아가로 오스 젤 전기 영동에 의해 아밀로이드 집계에 대한 심사

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

연구

생물학

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

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

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

유기 용매 및 초산없이 쿠매시 G - 250로 젤의 단백질 염색법

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

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

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

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

액상 복구와 분자량 기반 분류에 대한 GELFREE 8100 분획화 시스템을 사용하여

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

Improving the Success Rate of Protein Crystallization by Random Microseed Matrix Screening

Random microseed matrix screening (rMMS) is a protein crystallization technique in which seed crystals are added to random screens. By increasing the likelihood that crystals will grow in the metastable zone of a protein's phase diagram, extra crystallization leads are often obtained, the quality of crystals produced may be increased, and a good supply of crystals for data collection and soaking experiments is provided. Here we describe a general method for rMMS that may be applied to either sitting drop or hanging drop vapor diffusion experiments, established either by hand or using liquid handling robotics, in 96-well or 24-well tray format.

Here we describe a general method for random microseed matrix screening. This technique is shown to significantly increase the success rate of protein crystallization screening experiments, reduce the need for optimization, and provide a reliable supply of crystals for data collection and ligand-soaking experiments.

임의 Microseed 매트릭스 심사에 의해 단백질 결정화의 성공률을 향상

Random microseed matrix screening  (rMMS) is a protein crystallization technique in which seed crystals are added  to random screens. By increasing the ...

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

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

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

담배 원형질체 및 잎에 이분자 형광 보완에 의해 시각 단백질 - 단백질 상호 작용

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

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

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

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

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

막 단백질의 녹색 형광 단백질 기반의 표현 상영에<em&gt; 대장균</em

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

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

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

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

대변​​ 글루코 코르티코이드 분석 : equids는 비 침습적 부신 모니터링

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

Detection of Glycosaminoglycans by Polyacrylamide Gel Electrophoresis and Silver Staining

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

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

폴리아크릴아미드 젤 전기포및 은 염색에 의한 글리코아미노글리칸 검출

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

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

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

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

마우스의 점진적 저항 훈련을 위한 간단하고 저렴한 러닝 휠 모델

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

Simplifying angiogenesis studies with a modified matrix gel plug assay(수정된 매트릭스 겔 플러그 어세이를 통한 혈관신생 연구 간소화)

Modified In Vivo Matrix Gel Plug Assay for Angiogenesis Studies

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

저자 스포트라이트: Investigating Angiogenesis and Vessel Permeability Through a Modified Matrix Gel Plug Assay (수정된 매트릭스 겔 플러그 분석을 통한 혈관신생 및 혈관 투과성 조사)

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

혈관신생 연구를 위한 수정된 In Vivo Matrix Gel Plug Assay

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