이 작품은 싱가포르 학술 연구위원회 (ARC) [부여 번호 90/07]에 의해 지원되었다. 우리는 지원 Radiodurans 감사합니다.

이 실험 절차에 대한 완전하고 상세한 텍스트 프로토콜은 분자 생물학의 현재 프로토콜에서 사용할 수 있습니다. 겔 샌드위치의 조립 및 겔 장치에 대한 자세한 단계별 지침은 BioRad 웹사이트 3 확인할 수 있습니다. 필수 장비 : 유리 접시 (내부 및 외부) 10cm 셀 : 10.1 X 7.3 cm (내부 판), 10.1 X 8.2 cm (외부 판), BioRad 20cm 셀 : 20 X 20cm (내부 판), 20 X 22.3 cm (외부 판), BioRad 0.5-1.5 mm 겔 빗과 스페이서 겔 주조 스탠드 덮개와 케이블로 젤 장치 고전압 전원 공급 장치 난방 장치 블록이나 물 목욕 Serological pipettes 및 피펫 에이드 피펫 및 피펫 팁 겔 건조기 스캐너 시약 및 솔루션 : 요소 (ultrapure) 40 % polyacrylamide 솔루션 (29:1) 10 × TBE 솔루션 (트리스 - Borate, EDTA (에틸렌 다이아 민 테트라 초산) 버퍼) Deionized, 증류수 TEMED 30 % (W / V) 암모늄 persulfate 솔루션 0.5 X TBE 솔루션 포름 아미드 EDTA (에틸렌 다이아 민 테트라 초산) 크실렌 cyanol Bromphenol 블루 메탄올 에탄올 <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> 음량 </td><td colspan="3" style="text-align:center;"> 50 ML </td><td colspan="3" style="text-align:center;"> 60 ML </td><td colspan="3" style="text-align:center;"> 10 ML </td></tr><tr><td> 아크릴 아미드 농도 </td><td> 10% </td><td> 12.5 % </td><td> 15% </td><td> 10% </td><td> 12.5 % </td><td> 15% </td><td> 10% </td><td> 12.5 % </td><td> 15% </td></tr><tr><td> g 요소 </td><td> 24 </td><td> 24 </td><td> 24 </td><td> 28.8 </td><td> 28.8 </td><td> 28.8 </td><td> 4.8 </td><td> 4.8 </td><td> 4.8 </td></tr><tr><td> ML 40% 아크릴 (29:1) </td><td> 12.5 </td><td> 15.625 </td><td> 18.75 </td><td> 15 </td><td> 18.75 </td><td> 22.5 </td><td> 2.5 </td><td> 3.125 </td><td> 3.75 </td></tr><tr><td> UL 30 % APS </td><td> 166 </td><td> 166 </td><td> 166 </td><td> 199.2 </td><td> 199.2 </td><td> 199.2 </td><td> 33 </td><td> 33 </td><td> 33 </td></tr><tr><td> UL TEMED </td><td> 20 </td><td> 20 </td><td> 20 </td><td> 24 </td><td> 24 </td><td> 24 </td><td> 4 </td><td> 4 </td><td> 4 </td></tr><tr><td> 10 × TBE </td><td> 5 </td><td> 5 </td><td> 5 </td><td> 6 </td><td> 6 </td><td> 6 </td><td> 1 </td><td> 1 </td><td> 1 </td></tr><tr><td colspan="10"> deionized, 증류수로 볼륨을 채워 </td></tr></tbody></table> 표 1 : 단일 좌초 oligonucleotides를 해결하기위한 다양한 아크릴 아미드의 농도와 볼륨에 필요한 시약 및 솔루션 겔 샌드위치 어셈블리 및 젤 준비 <ol><li> 제조 업체의 설명에 따라 겔 조립 및 겔 - 캐스팅 챔버 3 겔을 수정. 0.5-1.5 mm 두께 스페이서를 사용합니다. </li><li> 분자 생물학의 현재 프로토콜에 따라 적절한 polyacrylamide 솔루션을 준비하거나 표 1에 나열. 20cm의 denaturing 아크릴 아미드 젤에 대한 X 22cm X 1.5 mm, 젤 솔루션 60 ML과 10.1 X 8.2 cm에 대한 X 1mm 겔 5 ML 젤 솔루션은 충분합니다. 예상 제품 / 밴드 몇 기지의 범위 내에있는 경우 큰 젤을 사용하는 다음, 더 이상 젤는 단일 염기의 차이로 밴드를 해결할 수 있습니다. 예상 밴드는 10 개 이상의 세포핵에 차이가있다면, 작은 젤은 조각을 해결하기 위해 적합한 것입니다. 귀하의 분리 요구 사항을 맞는 polyacrylamide의 비율을 선택, 높은 polyacrylamide의 농도는 낮은 분자량의 조각을 해결할 수 있습니다. 아크릴 아미드의 원하는 양의 ultrapure의 요소와 믹스를 사용합니다. 0.5-1 X TBE의 최종 농도를하고 deionized, 증류수로 볼륨을 채울 수있는 겔 믹스에 TBE 버퍼를 추가합니다. 전자렌지에 20 초 동안 솔루션을 가열하고 그것을 부드럽게 섞는다. 솔루션 손이 따뜻한 때까지 큰 겔 볼륨이 단계를 반복하십시오. </li><li> 즉시 serological 피펫과 두 개의 유리 판 사이에 자동 피펫 에이드를 사용하여 겔 하거라. 공기 방울을 도입하지 마십시오. 빗를 삽입하고 젤 30-60분을위한 중합 보자. </li></ol> 전기 영동 장치를 설정하고 젤 prerun <ol><li> 지침 3 제조에 따라 주조 챔버와 겔 장치에 어셈블리를에서 젤 분리합니다. </li><li> 유리 플레이트가 서브됩니다 낮은 버퍼 챔버 재치 실행 버퍼 (0.5-1 X TBE) 작성버퍼 2~3cm을 통합. 버퍼를 실행하는 젤의 맨 위쪽 버퍼 챔버를 입력합니다. </li><li> 조심스럽게 빗를 제거하고 도움말을 읽어 피펫 및 젤을 사용하여 실행 버퍼로 우물을 씻어. </li><li> 고전압 전원 공급 장치에 케이블에서 겔 시스템 및 플러그의 뚜껑을 연결합니다. 당신이 샘플을 로드할 수 있습니다 전에 젤을 가열하고 젤 남아 요소를 제거하려면 최소 30 분 동안 젤 prerun해야합니다. 최적 온도는 45-55 사이 여야합니다 ° C. 60보다 높은 온도 ° C 밴드는 얼룩이나 유리 조각이 균열있을 수 있으므로.을 피하십시오 prerun (겔 당 15-25 W)에 대한 지속적인 와트를 선택합니다. </li></ol> 샘플 준비 <ol><li> 그 동안에 샘플을 준비합니다. 따라서, 샘플 2 X 젤 로딩 믹스의 해당 금액을 추가합니다. 로딩 혼합은 일반적으로 90 % 포름 아미드, 0.5 % EDTA (에틸렌 다이아 민 테트라 초산), 0.1 % 크실렌 cyanol와 0.1 % bromphenol 파란색이 포함되어 있습니다. </li><li> 샘플이 젤에로드하기 전에 샘플을 몇 분 동안 70-90 ° C 사이의 샘플을 가열하여 열기가 변성해야합니다. </li></ol> 젤로드 및 실행 <ol><li> prerun가 완료되면 뚜껑을 제거하고 완전히 같은 요소가 우물에 leached 것처럼 언급한 주머니를 씻어. </li><li> 주머니 바닥에서 신중하게 샘플을 적용합니다. 공기 방울을 도입하지 마십시오. 버퍼를 로딩하면 실행되는 동안 같은 상태를 유지하기 위해 빈 주머니에 적용되어야합니다. </li><li> 뚜껑을 조립하고 55 젤 온도를 유지하기 위해 지속적인 와트에서 젤을 실행 ° C prerun과 유사합니다. 염료 앞에는 젤의 하단에 도달 때까지 마커 염료의 마이 그 레이션을 관찰합니다. 운영 기간은 사용되는 아크릴 아미드의 비율, 버퍼와 겔 두께의 이온 강도에 따라 좌우됩니다. 실행 2-4시간 사이에 마지막으로하실 수 있습니다. 젤의 온도를 확인합니다. 겔 온도계가 실행되는 동안 적당한 온도를 모니터링하는 데 도움이 될 수 있습니다. </li></ol> 젤 공정 <ol><li> 염료 앞에는 젤의 끝에 도달하면 낮은 버퍼 챔버의 겔을 넣어. 챔버에서 젤을 제거하고 클램프를 느슨하게. 스페이서 멀리 당겨 조심스럽게 유리 접시를 감추고 있기도 하죠. 필요한 잘 겔 부분을 포함하고있는 상위 버려야하는 경우. </li><li> 조심스럽게 겔 고정 솔루션 (TBE 플러스 5-10%의 메탄올과 에탄올의 농도가 동일)와 함께 접시에 젤을 전송하기만하면됩니다. 50~10분위한 솔루션으로 젤두고 버퍼를 두 번 변경할 수 있습니다. </li><li> 젤은 진공 겔 건조기 또는 젤 직접 스캔을 사용하여 추가 처리를위한 준비가되었습니다. </li></ol>

<ol>
	<li>Maniatis, T., Jeffrey, A., van deSande, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chain+length+determination+of+small+double-+and+single-stranded+DNA+molecules+by+polyacrylamide+gel+electrophoresis.">Chain length determination of small double- and single-stranded DNA molecules by polyacrylamide gel electrophoresis.</a> Biochemistry. 14, 3787-3794 (1975).</li><li>Albright, L. M., Slatko, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Denaturing+polyacrylamide+gel+electrophoresis.">Denaturing polyacrylamide gel electrophoresis.</a> Curr Protoc Nucleic Acid Chem. , (2001).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> PROTEAN II xi Cell and PROTEAN II xi 2-D Cell Instruction Manual. , (2001).</li></ol>

The authors have nothing to disclose.

<ol><li> 밴드 날카로움은 여러 가지 요인에 따라 달라집니다. 선명한 밴드를위한로드 샘플의 볼륨 즉, 이상적으로 1-5 μl 사이에 가능한 작게해야합니다. 그러나, 최대 15 μl 여전히 허용 해상도를 보장하는 로드할 수 있습니다. 선명 밴드의 적은 샘플 량 결과. </li><li> 밴드 품질 또한 겔의 두께에 따라 좌우됩니다. 같은 0.75 mm로 얇은 젤은, 1.5 mm 두께 젤보다 더 나은 결과를 보여줍니다. </li><li> 샘플은 겔 주머니에 동일...

denaturing urea polyacrylamide gel electrophoresis (urea page)

요소 페이지 또는 요소의 polyacrylamide 젤 전기 영동을 denaturing는 보조 DNA 또는 RNA의 구조를 denatures 및 분자량에 따라 polyacrylamide 젤 매트릭스에서 분리에 사용되는 6-8 M의 요소를 고용합니다. 2-500 기지 사이의 조각은 단일 염기만큼 작은 길이의 차이로이 방법 1을 사용하여 분리 수 있습니다. 샘플의 마이 그 레이션 선택한 아크릴 아미드의 농도에 따라 좌우됩니다. polyacrylamide의 높은 비율의 낮은 분자량 조각을 해결합니다. 요소와 45-55의 온도의 조합 ° C 젤 실행하는 동안 비정형 DNA 또는 RNA 분자의 분리 수 있습니다. 일반적으로이 방법은 단일 좌초된 DNA 또는 효소 절단 반응에서 합성 또는 분류 oligonucleotides 또는 제품으로 RNA 조각을 분석하거나 정화가 필요합니다. 이 비디오를 문서에서 우리는 denaturing 요소의 polyacrylamide의 젤류을 준비하고 실행하는 방법을 보여줍니다. 기술 팁은 원래 프로토콜 1,2 이외에 포함되어 있습니다.

Watch this Scientific Journal Video about Denaturing Urea Polyacrylamide Gel Electrophoresis Urea PAGE at JoVE.com

Denaturing Urea Polyacrylamide Gel Electrophoresis Urea PAGE

Denaturing 요소의 polyacrylamide 젤 전기 영동은 단일 좌초 DNA를 분리 또는 500 세포핵의 한계까지 RNA하는 데 사용됩니다. 열 denatures 샘플 및 구조화되지 않은 단일 가닥와 함께 요소는 분자량에 따라 겔 매트릭스 내에서 마이 그 레이션.

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 ...

denaturing-urea-polyacrylamide-gel-electrophoresis-urea-page

Research

JoVE Journal

Biology

Denaturing Urea Polyacrylamide Gel Electrophoresis (Urea PAGE)

78.3K 조회수.  Nanyang Technological University, Singapore - NTU. Denaturing 요소의 polyacrylamide 젤 전기 영동은 단일 좌초 DNA를 분리 또는 500 세포핵의 한계까지 RNA하는 데 사용됩니다. 열 denatures 샘플 및 구조화되지 않은 단일 가닥와 함께 요소는 분자량에 따라 겔 매트릭스 내에서 마이 그 레이션.

비디오: Denaturing Urea Polyacrylamide Gel Electrophoresis Urea PAGE

Denaturing Gradient Gel Electrophoresis (DGGE)

Denaturing Gradient Gel Electrophoresis DGGE

Denaturing 그라디언트 젤 전기 영동 (DGGE)

연구

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 ...

Fast and Sensitive Colloidal Coomassie G-250 Staining for Proteins in Polyacrylamide Gels

Coomassie Brilliant Blue (CBB) is a dye commonly used for the visualization of proteins separated by SDS-PAGE, offering a simple staining procedure and high quantitation. Furthermore, it is completely compatible with mass spectrometric protein identification. But despite these advantages, CBB is regarded to be less sensitive than silver or fluorescence stainings and therefore rarely used for the detection of proteins in analytical gel-based proteomic approaches.
Several improvements of the original Coomassie protocol1 have been made to increase the sensitivity of CBB. Two major modifications were introduced to enhance the detection of low-abundant proteins by converting the dye molecules into colloidal particles: In 1988, Neuhoff and colleagues applied 20% methanol and higher concentrations of ammonium sulfate into the CBB G-250 based staining solution2, and in 2004 Candiano et al. established Blue Silver using CBB G-250 with phosphoric acid in the presence of ammonium sulfate and methanol3. Nevertheless, all these modifications just allow a detection of approximately 10 ng protein. A widely fameless protocol for colloidal Coomassie staining was published by Kang et al. in 2002 where they modified Neuhoff's colloidal CBB staining protocol regarding the complexing substances. Instead of ammonium sulfate they used aluminum sulfate and methanol was replaced by the less toxic ethanol4. The novel aluminum-based staining in Kang's study showed superior sensitivity that detects as low as 1 ng/band (phosphorylase b) with little sensitivity variation depending on proteins.
Here, we demonstrate application of Kang's protocol for fast and sensitive colloidal Coomassie staining of proteins in analytical purposes. We will illustrate the quick and easy protocol using two-dimensional gels routinely performed in our working group.

This video shall popularize a colloidal Coomassie G-250 staining protocol according to Kang et al. for the detection of average 4 ng protein in gels. The staining is completed within 2 hours and without any effort. We routinely use Kang's protocol for analytical purposes in gel-based proteomics.

Polyacrylamide의 젤의 단백질에 대한 신속하고 민감한 콜로이드 쿠매시 G - 250 스테 이닝

Coomassie Brilliant Blue (CBB) is a dye commonly used for the visualization of proteins separated by SDS-PAGE, offering a simple staining procedure and ...

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

Multiprotein complexes (MPCs) play a crucial role in cell signalling, since most proteins can be found in functional or regulatory complexes with other proteins (Sali, Glaeser et al. 2003). Thus, the study of protein-protein interaction networks requires the detailed characterization of MPCs to gain an integrative understanding of protein function and regulation. For identification and analysis, MPCs must be separated under native conditions. In this video, we describe the analysis of MPCs by blue native polyacrylamide gel electrophoresis (BN-PAGE). BN-PAGE is a technique that allows separation of MPCs in a native conformation with a higher resolution than offered by gel filtration or sucrose density ultracentrifugation, and is therefore useful to determine MPC size, composition, and relative abundance (Sch&auml;gger and von Jagow 1991); (Sch&auml;gger, Cramer et al. 1994). By this method, proteins are separated according to their hydrodynamic size and shape in a polyacrylamide matrix. Here, we demonstrate the analysis of MPCs of total cellular lysates, pointing out that lysate dialysis is the crucial step to make BN-PAGE applicable to these biological samples. Using a combination of first dimension BN- and second dimension SDS-PAGE, we show that MPCs separated by BN-PAGE can be further subdivided into their individual constituents by SDS-PAGE. Visualization of the MPC components upon gel separation is performed by standard immunoblotting. As an example for MPC analysis by BN-PAGE, we chose the well-characterized eukaryotic 19S, 20S, and 26S proteasomes.

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

In this video, we describe the characterization of multiprotein complexes (MPCs) by blue native polyacrylamide gel electrophoresis (BN-PAGE). In a first dimension, dialyzed cellular lysates are separated by BN-PAGE to identify individual MPCs. In a second dimension SDS-PAGE, MPCs of interest are further subdivided to analyze their constituents by immunoblotting.

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 ...

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 ...

Preparation of DNA-crosslinked Polyacrylamide Hydrogels

Mechanobiology is an emerging scientific area that addresses the critical role of physical cues in directing cell morphology and function. For example, the effect of tissue elasticity on cell function is a major area of mechanobiology research because tissue stiffness modulates with disease, development, and injury. Static tissue-mimicking materials, or materials that cannot alter stiffness once cells are plated, are predominately used to investigate the effects of tissue stiffness on cell functions. While information gathered from static studies is valuable, these studies are not indicative of the dynamic nature of the cellular microenvironment in vivo. To better address the effects of dynamic stiffness on cell function, we developed a DNA-crosslinked polyacrylamide hydrogel system (DNA gels). Unlike other dynamic substrates, DNA gels have the ability to decrease or increase in stiffness after fabrication without stimuli. DNA gels consist of DNA crosslinks that are polymerized into a polyacrylamide backbone. Adding and removing crosslinks via delivery of single-stranded DNA allows temporal, spatial, and reversible control of gel elasticity. We have shown in previous reports that dynamic modulation of DNA gel elasticity influences fibroblast and neuron behavior. In this report and video, we provide a schematic that describes the DNA gel crosslinking mechanisms and step-by-step instructions on the preparation DNA gels.

Our laboratory has developed DNA-crosslinked polyacrylamide hydrogels, a dynamic hydrogel system, to better understand the effects of modulating tissue stiffness on cell function. Here, we provide schematics, descriptions, and protocols to prepare these hydrogels.

DNA-가교 폴리 아크릴 아미드 하이드로 겔의 제조

Mechanobiology is an emerging scientific area that addresses the critical role of physical cues in directing cell morphology and function. For example, ...

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 ...

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 ...

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 ...

Dissecting, Fixing, and Visualizing the Drosophila Pupal Notum

The pupae of Drosophila melanogaster are immobile for several days during metamorphosis, during which they develop a new body with a thin transparent adult integument. Their immobility and transparency make them ideal for in vivo live imaging experiments. Many studies have focused on the dorsal epithelial monolayer of the pupal&#160;notum because of its accessibility and relatively large size. In addition to the studies of epithelial mechanics and development, the notum has been an ideal tissue to study wound healing. After an injury, the entire epithelial repair process can be captured by live imaging over 6-12 h. Despite the popularity of the notum for live imaging, very few published studies have utilized fixed notum samples. Fixation and staining are common approaches for nearly all other Drosophila tissues, taking advantage of the large repertoire of simple cellular stains and antibodies. However, the pupal notum is fragile and prone to curling and distortion after removal from the body, making it challenging to complement live imaging. This protocol offers a straightforward method for fixing and staining the pupal notum, both intact and after laser-wounding. With this technique, the ventral side of the pupa is glued down to a coverslip to immobilize the pupa, and the notum is carefully removed, fixed, and stained. The notum epithelium is mounted on a slide or between two coverslips to facilitate imaging from the tissue&#39;s dorsal or ventral side.

Dissecting, Fixing, and Visualizing the Drosophila Pupal Notum

The present protocol details the preparation and visualization of fixed tissue of the Drosophila pupal notum. It can be used for either intact or wounded tissue, and the original architecture of the tissue is preserved. The procedures for dissecting, fixing, and staining are all described in this article.

Drosophila Pupal Notum 해부, 고정 및 시각화

The pupae of Drosophila melanogaster are immobile for several days during metamorphosis, during which they develop a new body with a thin transparent ...