우리는 마이클 Reth, 헤르만 Schägger와 마가리타 과학 지원 카마초 - Carvajal 감사합니다. 이 작품은 독일 Forschungsgemeinschaft (DFG)에서 BIOSS하여 Bundesministerium fuer Bildung 및 Forschung (BMBF)에서 FO​​RSYS에 의해 재정 지원하고, 독일 연방 및 주 정부의 우수 이니셔티브 (GSC - 4, Spemann 대학원에서 일부 지원이 ).

<ol>
	<li>Arnold, I., Pfeiffer, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Yeast+mitochondrial+F1F0-ATP+synthase+exists+as+a+dimer:+identification+of+three+dimer-specific+subunits.">Yeast mitochondrial F1F0-ATP synthase exists as a dimer: identification of three dimer-specific subunits.</a> EMBO J. 17 (24), 7170-7178 (1998).</li><li>Camacho-Carvajal, M. M., Wollscheid, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-dimensional+Blue+native/SDS+gel+electrophoresis+of+multi-protein+complexes+from+whole+cellular+lysates:+a+proteomics+approach.">Two-dimensional Blue native/SDS gel electrophoresis of multi-protein complexes from whole cellular lysates: a proteomics approach.</a> Mol. Cell. Proteomics. 3 (2), 176-182 (2004).</li><li>Heiss, K., Junkes, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subproteomic+analysis+of+metal-interacting+proteins+in+human+B+cells.">Subproteomic analysis of metal-interacting proteins in human B cells.</a> Proteomics. 5 (14), 3614-3622 (2005).</li><li>Sali, A., Glaeser, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+words+to+literature+in+structural+proteomics.">From words to literature in structural proteomics.</a> Nature. 422, 216-225 (2003).</li><li>Schägger, H., Cramer, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+molecular+masses+and+oligomeric+states+of+protein+complexes+by+blue+native+electrophoresis+and+isolation+of+membrane+protein+complexes+by+two-dimensional+native+electrophoresis.">Analysis of molecular masses and oligomeric states of protein complexes by blue native electrophoresis and isolation of membrane protein complexes by two-dimensional native electrophoresis.</a> Anal. Biochem. 217 (2), 220-230 (1994).</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=Blue+native+electrophoresis+for+isolation+of+membrane+protein+complexes+in+enzymatically+active+form.">Blue native electrophoresis for isolation of membrane protein complexes in enzymatically active form.</a> Anal. Biochem. 199 (2), 223-231 (1991).</li><li>Schamel, W. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biotinylation+of+protein+complexes+may+lead+to+aggregation+as+well+as+to+loss+of+subunits+as+revealed+by+Blue+Native+PAGE.">Biotinylation of protein complexes may lead to aggregation as well as to loss of subunits as revealed by Blue Native PAGE.</a> J Immunol Methods. 252 (1-2), 171-174 (2001).</li><li>Schamel, W. W., Arechaga, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coexistence+of+multivalent+and+monovalent+TCRs+explains+high+sensitivity+and+wide+range+of+response.">Coexistence of multivalent and monovalent TCRs explains high sensitivity and wide range of response.</a> J. Exp. Med. 202, 493-503 (2005).</li><li>Schamel, W. W., Reth, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monomeric+and+oligomeric+complexes+of+the+B+cell+antigen+receptor.">Monomeric and oligomeric complexes of the B cell antigen receptor.</a> Immunity. 13 (1), 5-14 (2000).</li><li>Swamy, M., Minguet, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+native+antibody-based+mobility-shift+technique+(NAMOS-assay)+to+determine+the+stoichiometry+of+multiprotein+complexes.">A native antibody-based mobility-shift technique (NAMOS-assay) to determine the stoichiometry of multiprotein complexes.</a> J Immunol Methods. 324 (1-2), 74-83 (2007).</li><li>Swamy, M., Siegers, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blue+native+polyacrylamide+gel+electrophoresis+(BN-PAGE)+for+the+identification+and+analysis+of+multiprotein+complexes.">Blue native polyacrylamide gel electrophoresis (BN-PAGE) for the identification and analysis of multiprotein complexes.</a> Sci STKE. 2006 (345), 4-4 (2006).</li><li>Wittig, I., Schagger, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Features+and+applications+of+blue-native+and+clear-native+electrophoresis.">Features and applications of blue-native and clear-native electrophoresis.</a> Proteomics. 8, 3974-3990 (2008).</li></ol>

관심 없음 충돌 선언하지 않습니다.

본 연구에서는, 우리는 BN - PAGE에 의해 MPCs의 분석을 설명합니다. 2D 접근이 기본 조건 첫째 별도 MPCs하는 데 사용됩니다, 그리고 추가로 두 번째 차원 SDS - PAGE하여 개별 구성 요소로 그들을 나눌 수 있습니다. 샘플은 세포 lysates의 준비가되어 있습니다. 많은 MPCs의 solubilization 들어, 적절한 세제는 단백질 단지의 구조를 유지하는, 필요합니다. 여기, 우리는 0.1 % 트리톤 X - 100을 사용합니다. 그러나, 최적의 세제와 적당한 농도는 모든 MPC에 대해 경험적으로 결정해야합니다. 의 경우에는 트리톤 X - 100, 예를 들면, 그것은 낮은 세제 농도가 F 1 dimeric 양식의 식별을 허용되는 것으로보고되었습니다 F 0 - ATPase 복합 (아놀드, 파이퍼 외. 1998). 높은 트리톤 X - 100 농도는하지만, 이합체의 분리와 monomeric F 1 F 0 - ATPase 복합 해당 증가로 이어집니다. 높은 농도 또는 다른 세제의 사용가 digitonin 결과를 불리는 반면, 이것은 과거의 연구 중 하나와 라인에, 우리는 Brij 96 낮은 농도로 추출하면 multivalent T - 세포 수용체 복합 (TCR)가 보존는 것을 보여주고 있었다 monomeric TCR의 추출 (Schamel, Arechaga 외. 2005). 테스트할 수 있습니다 일반적으로 사용되는 세제는 digitonin (0.5-1 %), 트리톤 X - 100 (0.1-0.5 %), Brij 96 (0.1-0.5 %), 또는 dodecylmaltoside을 (0.1-0.5 %) 등이 있습니다. 이 시약은 MPC의 안정성을 위해 최선이 될 경향이 nonionic 세제입니다. SDS와 다른 강한 세제과 접촉이 (카마초 - Carvajal, Wollscheid 외. 2004) 피해야한다고 알고 있습니다. lysates의 투석은 BN - 젤의 MPC 분리 (. 카마초 - Carvajal, Wollscheid 외 2004) 달성하는 데 필요한, (Heiss, Junkes 외 2005.). 그것은 소금 농도 또는 낮은 분자량의 불순물의 제거의 조정은 높은 해상도를 위해 중요 것 같습니다. 또한 immunopurified 나중에 항체에서 eluted되었습니다 막 준비와 MPCs이 (스와미, Siegers 외. 2006) BN - PAGE에 적합한 것을 주목할만한 것입니다. 두 경우 모두에서, 샘플은 멤브레인 용해 또는 용출이 BN - 용해 버퍼에서 실시하는 경우, BN - PAGE 분리에 대한 dialyzed 될 필요가 없습니다. BN - PAGE로 단백질 분리, 염료 쿠매시 파란색 단백질 unspecifically 바인딩과 부정 청구와 ​​함께 그들을 포함하는, 필요합니다. 따라서, 쿠매시 파란색 중립 PH (Schägger 및 본 Jagow 1991)에서 음극쪽으로 단백질의 전기 영 동적 이동성을 가능하게, (Schägger, 크래머 외 1994.). 또한, 쿠매시 파란색 전기 영동하는 동안 쌓아 겔의 단백질 집합을 방지합니다. BN - PAGE 들어, 쿠매시 G250은 쿠매시 청색 R250이나 콜로이드 쿠매시 블루스 대신 사용할 수 있습니다. BN - 젤을 실행하기 전에, 그것은 젤의 비율은 관심의 MPC의 예상 크기에 맞는 것을 보장하기 위해 필요합니다. 다양한 그라디언트와 적절한 버퍼 Invitrogen (NativePAGE Novex BIS - 트리스 젤 시스템)에서 상업적으로 사용할 수로 BN - 젤을 프리 캐스트. 그러나 BN - 젤 또한 persistaltic 펌프와 함께 그라디언트 믹서를 사용하여 준비하실 수 있습니다. 손상 그라데이션을 보장하기 위해 액체가 쏟아져과 거품을 피해야한다 동안 끊임없이 유동한다. 과부하가 전기 과정 중에 단백질 침전으로 이어질 수 있기 때문에 우리는 겔에 다른 샘플 dilutions의 로딩을 권장합니다. 또한, BN - 젤은 4 실행되어야 ° C 단백질 저하를 방지하고 MPCs 그대로 유지합니다. MPCs의 BN - PAGE, 시각화가 쿠매시 빛나는 푸른 얼룩, 실버 얼룩이나 immunoblotting하여 얻을 수있다 후에. 단백질 밴드 대량 분석계 (카마초 - Carvajal, Wollscheid 외. 2004)에 의해 더 분석에 적합합니다 쿠매시 또는 실버 얼룩의 시각. immunoblotting의 경우 관심의 MPCs에 대한 최적의 전송 상태는 경험적으로 결정해야합니다. 쿠매시 블루도 BN - 젤의 모래 바닥 동안 전송되는 점에 유의하십시오. 막이 파란 색깔을 발휘합니다 반면 따라서, 젤은 성공적인 전송 후에 무색됩니다. 또한, SDS - PAGE 후 검색을 위해 일하는 게 아니 모든 기본 항체가, BN - PAGE에 immunoblotting에 적용할 수 있다고 언급하는 것이 중요합니다. 그것은 그들의 에피토프이 단백질의 원래 형태에 숨겨져 있기 때문에 항체가 관심의 MPC를 인식하지 않는 것이 발생할 수 있습니다. 이 문제를 극복하기 위해서는 1X SDS 샘플 버퍼에 곧 젤 끓는 사전 전송 BN - 겔 내에서 단백질을 변성 수 있습니다. 우리의 예제에서는, 우리는 단백질 밴드의 검출에 직접 BN - 젤 적용되지 않았다. 대신, 우리는 추가로 두 번째 dimensi하여 BN - PAGE로 구분 lysate를 나누어SDS - PAGE에. 두 번째 차원 SDS - 겔에서 monomeric 단백질은 두 번째 차원 (카마초 - Carvajal, Wollscheid 외. 2004)의 첫 번째와 선형 겔에서 그라디언트 젤로 인해 쌍곡선 대각선 이내에 마이 그 레이션. 그들이 쌍곡선 대각선 아래화된되기 때문에 이것은, MPCs의 쉽게 확인하실 수 있습니다. 한 별개의 MPC의 하위 구성 요소는 두 번째 차원 SDS - PAGE의 수직선에서 구분됩니다. 여러 dinstinct의 MPCs의 성분 구성 요소는 MPC의 크기에 따라 수평선에서 확인할 수 있습니다. 그러나, 하나의 수직선에 나타나는 여러 가지 단백질의 명소도 BN - PAGE의 동일한 위치에서 마이 그 레이션 개별 단지의 일부가 될 수 있다고 간주되어야한다. 그들은 같은 MPC에 나타날 수있는 마지막 증거는 항체 기반 겔 변화 분석에 의해 얻을 수 있습니다. 이 분석에서는 셀룰러 lysate 전에 BN - PAGE로 확인 명소 중 하나에 의해 대표되는 단백질에 대한 항체와 함께 incubated입니다. 첫 번째 차원 높은 분자 질량으로이 단백질을 포함하는 모든 MPCs의 변화이 발생합니다. 이러한 MPCs의 일부 다른 단백질이 복잡한 특정 변화를 받아야하므로 두 번째 차원 SDS - 겔에서 식별하는 쉬운 것입니다. 뿐만 아니라 MPCs의 구성은 BN - PAGE로 분석뿐만 아니라 자신의 stoichiometry의 결정은 (Schamel 및 Reth 2000) 수도 수 있으며 (Schamel 2001), (스와미, Minguet 외 2007.). 이를 위해 NAMOS 분석 (기본 항체 기반 이동성 교대 분석)을 수행할 수 있습니다. 항체 기반 겔 변화 분석에서와 마찬가지로, 세포 lysates은 단클론 subunit 특정 항체와 incubated 있습니다. 이것은 MPCs의 stoichiometry에있는 변화의 범위에서 추론을 허용 BN - 젤에서 전기 immunoshifts의 유도에 연결 conlusion에서 BN - PAGE은 MPCs의 식별과 크기, 구성뿐만 아니라, 상대 풍요의 결정에 적합합니다. NAMOS 분석으로 수행, 그것은 또한 특정 MPC의 stoichiometry을 결정할 수있는 가능성을 제공합니다. 의 일반적인 적용을 감안할 때,이 기법은 MPCs의 특성 (. 데커, 뮐러 외 1996)에 대한 매우 유용한 도구입니다, (위티그 및 Schagger 2008), (바그너, Rehling 외 2009)., (위티그 및 Schägger 2009) .

blue native polyacrylamide gel electrophoresis (bn-page) for analysis of multiprotein complexes from cellular lysates

대부분의 단백질이 다른 단백질과 기능이나 규제 단지 (살리, Glaeser 외. 2003)에서 찾을 수 있으므로 Multiprotein의 단지 (MPCs)은 세포 신호 전달에 중요한 역할을한다. 따라서, 단백질 - 단백질 상호 작용 네트워크의 연구는 단백질 기능과 규정의 통합 이해를 얻을 수 MPCs의 세부 특성이 필요합니다. 식별 및 분석을 위해, MPCs는 기본 조건 구분해야합니다. 이 비디오에서는, 우리는 푸른 원시의 polyacrylamide 겔 전기 영동 (BN - PAGE)에 의해 MPCs의 분석을 설명합니다. BN - PAGE은 젤 여과 또는 자당 밀도 ultracentrifugation에 의해 제공되는보다 높은 해상도와 고유의 형태에 MPCs의 분리를 허용하고, MPC 크기, 구성, 그리고 상대적으로 풍부 (Schägger 및 본 Jagow 1991)를 결정하는 것이 유용합니다 기술입니다 ; (Schägger, 크래머 외 1994.). 이 방법으로 단백질들은 유체 크기와 polyacrylamide의 매트릭스에 형태에 따라 구분됩니다. 여기, 우리는 lysate 투석은 BN - PAGE 이러한 생물 학적 샘플에 적용을하는 중요한 단계입니다 지적, 전체 세포 lysates의 MPCs의 분석을 보여줍니다. 첫째 차원 BN -와 두 번째 차원 SDS - PAGE의 조합을 사용하여, 우리는 BN - PAGE로 구분 MPCs이 더욱 SDS - PAGE에 의해 그들의 개인적인 성분으로 세분화 될 수 보여줍니다. 겔 분리시 MPC 구성 요소의 시각화 표준 immunoblotting에 의해 수행됩니다. BN - PAGE에 의해 MPC 분석을위한 예를 들어, 우리는 잘 특징 진핵세포 19S, 20 대, 그리고 26S proteasomes를 선택했습니다.

Watch this Scientific Journal Video about Blue Native Polyacrylamide Gel Electrophoresis BN-PAGE for Analysis of Multiprotein Complexes from Cellular Lysates at JoVE.com

Blue Native Polyacrylamide Gel Electrophoresis BN-PAGE for Analysis of Multiprotein Complexes from Cellular Lysates

이 비디오에서는, 우리는 푸른 원시의 polyacrylamide 겔 전기 영동 (BN - PAGE)에 의해 multiprotein의 단지 (MPCs)의 특성을 설명합니다. 첫 번째 차원에서 dialyzed 세포 lysates은 개별 MPCs을 식별할 BN - PAGE로 구분됩니다. 두 번째 차원 SDS - PAGE에 관심 MPCs은 더욱 immunoblotting하여 성분을 분석하기 위해 세분화됩니다.

셀룰러 Lysates에서 Multiprotein의 단지의 분석을 위해 블루 기본 Polyacrylamide 젤 전기 영동 (BN - PAGE)

Multiprotein complexes (MPCs) play a crucial role in cell signalling, since most proteins can be found in functional or regulatory complexes with other ...

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Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) for Analysis of Multiprotein Complexes from Cellular Lysates

130.3K 조회수.  University of Freiburg. 이 비디오에서는, 우리는 푸른 원시의 polyacrylamide 겔 전기 영동 (BN - PAGE)에 의해 multiprotein의 단지 (MPCs)의 특성을 설명합니다. 첫 번째 차원에서 dialyzed 세포 lysates은 개별 MPCs을 식별할 BN - PAGE로 구분됩니다. 두 번째 차원 SDS - PAGE에 관심 MPCs은 더욱 immunoblotting하여 성분을 분석하기 위해 세분화됩니다.

비디오: Blue Native Polyacrylamide Gel Electrophoresis BN-PAGE for Analysis of Multiprotein Complexes from Cellular Lysates

Denaturing Gradient Gel Electrophoresis (DGGE)

Denaturing Gradient Gel Electrophoresis DGGE

Denaturing 그라디언트 젤 전기 영동 (DGGE)

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생물학

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 ...

Denaturing Urea Polyacrylamide Gel Electrophoresis (Urea PAGE)

Urea PAGE or denaturing urea polyacrylamide gel electrophoresis employs 6-8 M urea, which denatures secondary DNA or RNA structures and is used for their separation in a polyacrylamide gel matrix based on the molecular weight. Fragments between 2 to 500 bases, with length differences as small as a single nucleotide, can be separated using this method1. The migration of the sample is dependent on the chosen acrylamide concentration. A higher percentage of polyacrylamide resolves lower molecular weight fragments. The combination of urea and temperatures of 45-55 &deg;C during the gel run allows for the separation of unstructured DNA or RNA molecules.
In general this method is required to analyze or purify single stranded DNA or RNA fragments, such as synthesized or labeled oligonucleotides or products from enzymatic cleavage reactions.
In this video article we show how to prepare and run the denaturing urea polyacrylamide gels. Technical tips are included, in addition to the original protocol 1,2.

Denaturing Urea Polyacrylamide Gel Electrophoresis Urea PAGE

Denaturing urea polyacrylamide gel electrophoresis is used to separate single-stranded DNA or RNA up to a limit of 500 nucleotides. Urea in combination with heat denatures samples and unstructured single strands migrate within the gel matrix according to their molecular weight.

Denaturing 요소의 Polyacrylamide 젤 전기 영동 (우레아 페이지)

Urea PAGE or denaturing urea polyacrylamide gel electrophoresis employs 6-8 M urea, which denatures secondary DNA or RNA structures and is used for their ...

Agarose Gel Electrophoresis for the Separation of DNA Fragments

Agarose gel electrophoresis is the most effective way of separating DNA fragments of varying sizes ranging from 100 bp to 25 kb1. Agarose is isolated from the seaweed genera Gelidium and Gracilaria, and consists of repeated agarobiose (L- and D-galactose) subunits2. During gelation, agarose polymers associate non-covalently and form a network of bundles whose pore sizes determine a gel&#39;s molecular sieving properties. The use of agarose gel electrophoresis revolutionized the separation of DNA. Prior to the adoption of agarose gels, DNA was primarily separated using sucrose density gradient centrifugation, which only provided an approximation of size. To separate DNA using agarose gel electrophoresis, the DNA is loaded into pre-cast wells in the gel and a current applied. The phosphate backbone of the DNA (and RNA) molecule is negatively charged, therefore when placed in an electric field, DNA fragments will migrate to the positively charged anode. Because DNA has a uniform mass/charge ratio, DNA molecules are separated by size within an agarose gel in a pattern such that the distance traveled is inversely proportional to the log of its molecular weight3. The leading model for DNA movement through an agarose gel is &#34;biased reptation&#34;, whereby the leading edge moves forward and pulls the rest of the molecule along4. The rate of migration of a DNA molecule through a gel is determined by the following: 1) size of DNA molecule; 2) agarose concentration; 3) DNA conformation5; 4) voltage applied, 5) presence of ethidium bromide, 6) type of agarose and 7) electrophoresis buffer. After separation, the DNA molecules can be visualized under uv light after staining with an appropriate dye. By following this protocol, students should be able to: 
 
1. Understand the mechanism by which DNA fragments are separated within a gel matrix 
2. Understand how conformation of the DNA molecule will determine its mobility through a gel matrix 
3. Identify an agarose solution of appropriate concentration for their needs 
4. Prepare an agarose gel for electrophoresis of DNA samples 
5. Set up the gel electrophoresis apparatus and power supply 
6. Select an appropriate voltage for the separation of DNA fragments 
7. Understand the mechanism by which ethidium bromide allows for the visualization of DNA bands 
8. Determine the sizes of separated DNA fragments 
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A basic protocol for the separation of DNA fragments using agarose gel electrophoresis is described.

의 DNA 조각의 분리를위한 아가로 오스 젤 전기 영동

Agarose gel electrophoresis is the most effective way of separating DNA fragments of varying sizes ranging from 100 bp to 25 kb1. Agarose is isolated from ...

Rapid Generation of Amyloid from Native Proteins In vitro

Proteins carry out crucial tasks in organisms by exerting functions elicited from their specific three dimensional folds. Although the native structures of polypeptides fulfill many purposes, it is now recognized that most proteins can adopt an alternative assembly of beta-sheet rich amyloid. Insoluble amyloid fibrils are initially associated with multiple human ailments, but they are increasingly shown as functional players participating in various important cellular processes. In addition, amyloid deposited in patient tissues contains nonproteinaceous components, such as nucleic acids and glycosaminoglycans (GAGs). These cofactors can facilitate the formation of amyloid, resulting in the generation of different types of insoluble precipitates. By taking advantage of our understanding how proteins misfold via an intermediate stage of soluble amyloid precursor, we have devised a method to convert native proteins to amyloid fibrils in vitro. This approach allows one to prepare amyloid in large quantities, examine the properties of amyloid generated from specific proteins, and evaluate the structural changes accompanying the conversion.

Rapid Generation of Amyloid from Native Proteins In vitro

Proteins can either adopt a native structure or misfold into insoluble amyloid. Conditions that favor the misfolding pathway lead to the formation of different types of amyloid fibrils. The methods described here allow rapid conversion of native proteins into amyloid in vitro.

천연 단백질에서 아밀로이드의 빠른 생성<em&gt; 체외</em

Proteins carry out crucial tasks in organisms by exerting functions elicited from their specific three dimensional folds. Although the native structures ...

A New Approach for the Comparative Analysis of Multiprotein Complexes Based on 15N Metabolic Labeling and Quantitative Mass Spectrometry

The introduced protocol provides a tool for the analysis of multiprotein complexes in the thylakoid membrane, by revealing insights into complex composition under different conditions. In this protocol the approach is demonstrated by comparing the composition of the protein complex responsible for cyclic electron flow (CEF) in Chlamydomonas reinhardtii, isolated from genetically different strains. The procedure comprises the isolation of thylakoid membranes, followed by their separation into multiprotein complexes by sucrose density gradient centrifugation, SDS-PAGE, immunodetection and comparative, quantitative mass spectrometry (MS) based on differential metabolic labeling (14N/15N) of the analyzed strains. Detergent solubilized thylakoid membranes are loaded on sucrose density gradients at equal chlorophyll concentration. After ultracentrifugation, the gradients are separated into fractions, which are analyzed by mass-spectrometry based on equal volume. This approach allows the investigation of the composition within the gradient fractions and moreover to analyze the migration behavior of different proteins, especially focusing on ANR1, CAS, and PGRL1. Furthermore, this method is demonstrated by confirming the results with immunoblotting and additionally by supporting the findings from previous studies (the identification and PSI-dependent migration of proteins that were previously described to be part of the CEF-supercomplex such as PGRL1, FNR, and cyt f). Notably, this approach is applicable to address a broad range of questions for which this protocol can be adopted and e.g. used for comparative analyses of multiprotein complex composition isolated from distinct environmental conditions.

A New Approach for the Comparative Analysis of Multiprotein Complexes Based on 15N Metabolic Labeling and Quantitative Mass Spectrometry

The described comparative, quantitative proteomic approach aims at obtaining insights into the composition of multiprotein complexes&#160;under different conditions and is demonstrated by comparing genetically different strains. For quantitative analysis equal volumes of different fractions from a sucrose density gradient are mixed and analyzed by mass spectrometry.

multiprotein의 단지의 비교 분석을위한 새로운 접근 방식을 기반으로<sup&gt; 15</sup&gt; N 대사 라벨링 및 정량 질량 분석

The introduced protocol provides a tool for the analysis of multiprotein complexes in the thylakoid membrane, by revealing insights into complex ...

AFM-based Mapping of the Elastic Properties of Cell Walls: at Tissue, Cellular, and Subcellular Resolutions

We describe a recently developed method to measure mechanical properties of the surfaces of plant tissues using atomic force microscopy (AFM) micro/nano-indentations, for a JPK&#160;AFM. Specifically, in this protocol we measure the apparent Young&#8217;s modulus of cell walls at subcellular resolutions across regions of up to 100 &#181;m&#160;x 100 &#181;m in floral meristems, hypocotyls, and roots. This requires careful preparation of the sample, the correct selection of micro-indenters and indentation depths. To account for cell wall properties only, measurements are performed in highly concentrated solutions of mannitol in order to plasmolyze the cells and thus remove the contribution of cell turgor pressure.
In contrast to other extant techniques, by using different indenters and indentation depths, this method allows simultaneous multiscale measurements, i.e. at subcellular resolutions and across hundreds of cells comprising a tissue. This means that it is now possible&#160;to spatially-temporally characterize the changes that take place in the mechanical properties of cell walls during development, enabling these changes to be correlated with growth and differentiation. This represents a key step to understand how coordinated microscopic cellular changes bring about macroscopic morphogenetic events.
However, several limitations remain: the method can only be used on fairly small samples (around 100 &#181;m in diameter) and only on external tissues; the method is sensitive to tissue topography; it measures only certain aspects of the tissue&#8217;s complex mechanical properties. The technique is being developed rapidly and it is likely that most of these limitations will be resolved in the near future.

We describe a method to map mechanical properties of plant tissues using an atomic force microscope (AFM). We focus on how to record mechanical changes that take place in cell walls during plant development at wide-field mesoscale, enabling these changes to be correlated with growth and morphogenesis.

셀 벽의 탄성 특성의 AFM 기반의 매핑 : 조직, 세포 및 세포 이하의 해상도에서

We describe a recently developed method to measure mechanical properties of the surfaces of plant tissues using atomic force microscopy (AFM) ...

Mucin Agarose Gel Electrophoresis: Western Blotting for High-molecular-weight Glycoproteins

Mucins, the heavily-glycosylated proteins lining mucosal surfaces, have evolved as a key component of innate defense by protecting the epithelium against invading pathogens. The main role of these macromolecules is to facilitate particle trapping and clearance while promoting lubrication of the mucosa. During protein synthesis, mucins undergo intense O-glycosylation and multimerization, which dramatically increase the mass and size of these molecules. These post-translational modifications are critical for the viscoelastic properties of mucus. As a result of the complex biochemical and biophysical nature of these molecules, working with mucins provides many challenges that cannot be overcome by conventional protein analysis methods. For instance, their high-molecular-weight prevents electrophoretic migration via regular polyacrylamide gels and their sticky nature causes adhesion to experimental tubing. However, investigating the role of mucins in health (e.g., maintaining mucosal integrity) and disease (e.g., hyperconcentration, mucostasis, cancer) has recently gained interest and mucins are being investigated as a therapeutic target. A better understanding of the production and function of mucin macromolecules may lead to novel pharmaceutical approaches, e.g., inhibitors of mucin granule exocytosis and/or mucolytic agents. Therefore, consistent and reliable protocols to investigate mucin biology are critical for scientific advancement. Here, we describe conventional methods to separate mucin macromolecules by electrophoresis using an agarose gel, transfer protein into nitrocellulose membrane, and detect signal with mucin-specific antibodies as well as infrared fluorescent gel reader. These techniques are widely applicable to determine mucin quantitation, multimerization and to test the effects of pharmacological compounds on mucins.

Mucins are high-molecular-weight glycoconjugates, with size ranging from 0.2 to 200 megadalton (MDa). As a result of their size, mucins do not penetrate conventional polyacrylamide gels and require larger pores for separation. We provide a detailed protocol for mucin agarose gel electrophoresis to assess relative quantification and study polymer assembly.

점액 아가로 오스 겔 전기 영동 : 높은 분자량 당 단백질에 대한 웨스턴 블로 팅


Mucins, the heavily-glycosylated proteins lining mucosal surfaces, have evolved as a key component of innate defense by protecting the epithelium against ...

Purification of Native Complexes for Structural Study Using a Tandem Affinity Tag Method

Affinity purification approaches have been successful in isolating native complexes for proteomic characterization. Structural heterogeneity and a degree of compositional heterogeneity of a complex do not usually impede progress in conducting such studies. In contrast, a complex intended for structural characterization should be purified in a state that is both compositionally and structurally homogeneous as well as at a higher concentration than required for proteomics. Recently, there have been significant advances in the application of electron microscopy for structure determination of large macromolecular complexes. This has heightened interest in approaches to purify native complexes of sufficient quality and quantity for structural determination by electron microscopy. The Tandem Affinity Purification (TAP) method has been optimized to extract and purify an 18-subunit, ~ 0.8 MDa ribonucleoprotein assembly from budding yeast (Saccharomyces cerevisiae) suitable for negative stain and electron cryo microscopy. Herein is detailed the modifications made to the TAP method, the rationale for making these changes, and the approaches taken to assay for a compositionally and structurally homogeneous complex.

The Tandem Affinity Purification (TAP) method has been used extensively to isolate native complexes from cellular extract, primarily eukaryotic, for proteomics. Here, we present a TAP method protocol optimized for purification of native complexes for structural studies.

탠덤 선호도 태그 방법을 사용하여 구조 연구에 대한 기본 공단의 정화

Affinity purification approaches have been successful in isolating native complexes for proteomic characterization. Structural heterogeneity and a degree ...

Analysis of Mitochondrial Respiratory Chain Complexes in Cultured Human Cells using Blue Native Polyacrylamide Gel Electrophoresis and Immunoblotting

Mitochondrial respiration is performed by oxidative phosphorylation (OXPHOS) complexes within mitochondria. Internal and environmental factors can perturb the assembly and stability of OXPHOS complexes. This protocol describes the analysis of mitochondrial respiratory chain complexes by blue native polyacrylamide gel electrophoresis (BN-PAGE) in application to cultured human cells. First, mitochondria are extracted from the cells using digitonin, then using lauryl maltoside, the intact OXPHOS complexes are isolated from the mitochondrial membranes. The OXPHOS complexes are then resolved by gradient gel electrophoresis in the presence of the negatively charged dye, Coomassie blue, which prevents protein aggregation and ensures electrophoretic mobility of protein complexes towards the cathode. Finally, the OXPHOS complexes are detected by standard immunoblotting. Thus, BN-PAGE is a convenient and inexpensive technique that can be used to evaluate the assembly of entire OXPHOS complexes, in contrast to the basic SDS-PAGE allowing the study of only individual OXPHOS complex subunits.

This protocol demonstrates the analysis of mitochondrial respiratory chain complexes by blue native polyacrylamide gel electrophoresis. Here the method applied to cultured human cells is described.

경작 된 인간 세포 블루 네이티브 Polyacrylamide 젤 전기 이동 법 및 Immunoblotting 사용 하 여에서 미토 콘 드리 아 호흡 사슬 단지의 분석

Mitochondrial respiration is performed by oxidative phosphorylation (OXPHOS) complexes within mitochondria. Internal and environmental factors can perturb ...