نحن نشكر مايكل ريث ، Schägger هيرمان ، ومارغريتا كاماتشو - كارفاخال للحصول على الدعم العلمي. وقد تم تمويل هذا العمل من FORSYS fuer Bildung Bundesministerium والإضافية اطلعوا (BMBF) ، عن طريق BIOSS من جمعية الألمانية للبحوث (DFG) ، ومعتمدة في جزء من مبادرة التميز للحكومات الاتحادية والدولة الألمانية (GSC - 4 ، Spemann كلية الدراسات العليا ).

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	<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. 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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. 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الإعلان عن أي تضارب في المصالح.

في هذه الدراسة ، ونحن تصف تحليل البلدان المتوسطية الشريكة التي PAGE - BN. ويستخدم نهج 2D إلى البلدان المتوسطية الشريكة الأولى في ظل ظروف منفصلة الأصلي ، ومن ثم إلى مزيد من تقسيم منهم الى مكوناتها الفردية من جانب البعد الثاني SDS - PAGE. مستعدون lysates عينات من الخلايا. لكثير من البلدان المتوسطية الشريكة من solubilization ، هناك حاجة إلى المنظفات المناسبة ، مما يحافظ على بنية مجمعات البروتين. هنا ، فإننا نستخدم تريتون 0.1 ٪ X - 100. ومع ذلك ، فإن المنظفات الأمثل وتركيزه مناسبة ويتم تحديدها تجريبيا لكل لجنة السياسة النقدية. في حالة تريتون X - 100 ، على سبيل المثال ، فقد أفيد أن تركيزات منخفضة المنظفات تسمح بتحديد نموذج مثنوي من 1 واو واو 0 - أتباز المعقدة (أرنولد ، فايفر وآخرون 1998). أعلى تريتون X - 100 تركيزات ، ومع ذلك ، تؤدي إلى تفكك للثنائيات ، وإلى زيادة مماثلة في موحودي F 1 0 - F أتباز المعقدة. وهذا ينسجم مع إحدى دراساتنا السابقة ، وتبين لنا أن يتم الاحتفاظ متعددي الكفاءات المعقدة مستقبلات خلايا T (TCR) عندما انتزعت مع تركيزات منخفضة من بريج 96 ، في حين أن استخدام تركيز أعلى أو المنظفات آخر يطلق عليه النتائج في ديجيتونين استخراج TCR موحودي (Schamel ، خيمينيز وآخرون 2005). يشيع استخدام المنظفات التي يمكن اختبارها وتشمل ديجيتونين (0.5 إلى 1 ٪) ، تريتون X - 100 (0.1 إلى 0.5 ٪) ، بريج 96 (0.1 إلى 0.5 ٪) ، أو dodecylmaltoside (0.1 إلى 0.5 ٪). هذه الكواشف والمنظفات nonionic ، والتي تميل الى ان تكون الأفضل لاستقرار السياسة النقدية. أن تدرك أنه ينبغي تجنب الاتصال مع الحزب الديمقراطي الصربي والمنظفات القوية الأخرى (كاماتشو - كارفاخال ، Wollscheid آخرون 2004). مطلوب غسيل الكلى للجنة السياسة النقدية لتحقيق lysates فصل في هلام BN (كاماتشو - كارفاخال ، Wollscheid آخرون 2004) ؛ (Heiss ، Junkes وآخرون 2005). يبدو أن تعديل تركيز الملح أو إزالة الشوائب منخفضة الوزن الجزيئي أمر حاسم لدقة عالية. من الجدير بالذكر أيضا أن التحضيرات الغشاء والبلدان المتوسطية الشريكة ، والتي تم في وقت لاحق وimmunopurified eluted من الأضداد ، هي مناسبة لPAGE - BN (سوامي ، Siegers وآخرون 2006). في كلتا الحالتين ، فإن عينات لا يجب أن تكون لمدال BN - PAGE الانفصال ، وإذا نفذ تحلل الغشاء أو شطف في BN - تحلل العازلة. لفصل البروتين PAGE - BN ، هناك حاجة لCoomassie الصبغة الزرقاء ، والتي تربط unspecifically للبروتينات ، ويغطي عليها مع الشحنات السالبة. وبالتالي ، Coomassie الزرقاء تمكن التنقل الكهربي للبروتينات نحو الكاثود عند pH محايدة (وSchägger فون Jagow 1991) ، (Schägger ، كريمر وآخرون 1994). علاوة على ذلك ، Coomassie الزرقاء يمنع التجميع البروتين في هلام التراص خلال الكهربائي. لPAGE - BN ، Coomassie G250 يجب أن يكون استخدامها بدلا من الزرقاء Coomassie R250 أو الغروية Coomassie البلوز. قبل تشغيل BN - هلام ، فمن الضروري التأكد من أن النسبة المئوية للهلام يتلاءم مع الحجم المتوقع من لجنة السياسة النقدية في المصالح. BN - الجاهزة المواد الهلامية مع تدرجات مختلفة والمخازن المناسبة متوفرة تجاريا من Invitrogen (NativePAGE Novex النظام جل مكرر تريس). ولكن يمكن أيضا أن تكون المواد الهلامية BN - المعدة باستخدام خلاط التدرج مع مضخة persistaltic. لضمان وجود التدرج سليمة ، يجب أن تدفق السائل باستمرار خلال صب ويجب تجنب الفقاعات. نوصي تحميل التخفيفات عينة مختلفة على الجل بسبب الحمولة الزائدة يمكن ان يؤدي الى هطول الأمطار البروتين أثناء عملية الكهربائي. وبالإضافة إلى ذلك ، يمكن تشغيل المواد الهلامية في BN - 4 درجات مئوية لمنع تدهور البروتين والبلدان المتوسطية الشريكة للحفاظ على حالها. ويمكن أن يتحقق بعد BN - PAGE ، والتصور من البلدان المتوسطية الشريكة من خلال تلطيخ Coomassie الزرقاء الرائعة ، وتلطيخ الفضة أو immunoblotting. تصور العصابات البروتين تلطيخ Coomassie أو الفضة هي مناسبة لمزيد من التحليل عن طريق مطياف الكتلة (كاماتشو - كارفاخال ، Wollscheid وآخرون 2004). في حالة immunoblotting ، والظروف المثلى لنقل البلدان المتوسطية الشريكة من الفائدة يجب أن تكون مصممة تجريبيا. أن يدرك أيضا أن يتم نقل Coomassie زرقاء أثناء النشاف من هلام - BN. ولذلك ، سوف يكون عديم اللون الجل بعد نقل ناجحة ، في حين أن غشاء سيبذل اللون الأزرق. كذلك ، فإنه من المهم أن نذكر أن ليس كل الأجسام المضادة الأولية ، والتي تعمل للكشف عن بعد SDS - PAGE ، ينطبق على immunoblotting على PAGE - BN. يمكن أن يحدث أن الأجسام المضادة لا تعترف لجنة السياسة النقدية في المصالح لان مخفيا حاتمة بهم في التشكل الأصلية من البروتينات. للتغلب على هذه المشكلة ، فمن الممكن أن تفسد البروتينات داخل الجل - BN قبل نقل بالغلي الجل قريبا في المخزن 1X عينة SDS. في مثالنا ، نحن لم تخضع للBN جل مباشرة إلى الكشف عن عصابات من البروتين. بدلا من ذلك ، ونحن كذلك تنقسم إلى BN - PAGE فصل lysate بواسطة dimensi secondعلى SDS - PAGE. في البعد الثاني جل SDS ، والبروتينات داخل موحودي ترحيل قطري القطعي بسبب الجل التدرج في الأول وهلام الخطية في البعد الثاني (كاماتشو - كارفاخال ، Wollscheid وآخرون 2004). هذا يسمح للبلدان المتوسطية الشريكة من السهل تحديد ، لأن مترجم انهم تحت هذا قطري القطعي. يتم فصل المكونات الفرعية للجنة السياسة النقدية المتميزة واحد في خط عمودي في البعد الثاني SDS - PAGE. يمكن التعرف على المكونات التي يتم المكونة من البلدان المتوسطية الشريكة dinstinct عدة على خط أفقي وفقا لحجم لجنة السياسة النقدية. ومع ذلك ، فإنه ينبغي النظر إلى أن العديد من البقع التي تظهر البروتين في سطر واحد عمودي يمكن أيضا أن تكون جزءا من مجمعات منفصلة التي تهاجر في نفس الموضع في PAGE - BN. يمكن الحصول على الدليل الأخير على أن وجودهم في لجنة السياسة النقدية من قبل نفس مقايسة الأضداد هلام تحول مقرا لها. في هذا الاختبار ، يتم تحضين الخلوية lysate مع جسم مضاد ضد بروتين ممثلة واحدة من النقاط التي تم تحديدها قبل PAGE - BN. هذه النتائج في التحول من جميع البلدان المتوسطية الشريكة التي تحتوي على هذا البروتين نحو كتلة الجزيئية أعلى في البعد الأول. والبروتينات الأخرى التي هي أيضا جزء من هذه البلدان المتوسطية الشريكة الخضوع لهذا التحول معقدة محددة ، وبالتالي من السهل تحديد في البعد الثاني SDS - هلام. لا يمكن إلا أن يكون تحليل تركيبة البلدان المتوسطية الشريكة التي PAGE - BN ، بل أيضا في تحديد رياضيات الكيمياء من الممكن (وريث Schamel 2000) ؛ (Schamel 2001) ، (سوامي ، Minguet وآخرون 2007). لهذا الغرض ، يمكن إجراء فحص NAMOS (اللغة الأم المستندة إلى الضد التنقل التحول مقايسة). كما هو الحال في جل مقايسة الأضداد القائم على التحول ، ويتم تحضين lysates الخليوي مع الأجسام المضادة وحيدة النسيلة محددة الوحيدات. هذا يؤدي إلى تحريض immunoshifts الكهربي في المواد الهلامية BN ، التي تسمح للاستدلال عن مدى التحول في البلدان المتوسطية الشريكة للرياضيات الكيمياء في conlusion ، BN - PAGE مناسبة للتعرف على البلدان المتوسطية الشريكة ، وتحديد تكوينها ، والحجم ، فضلا عن وفرة نسبية. كما أجريت مقايسة NAMOS ، فإنه يوفر أيضا إمكانية لتحديد السياسة النقدية للرياضيات الكيمياء معينة. نظرا تطبيقه عام ، هذه التقنية هي أداة مفيدة جدا لتحديد خصائص البلدان المتوسطية الشريكة (ديكر ، مولر وآخرون 1996) ؛ (Wittig وSchagger 2008) ، (واغنر ، Rehling وآخرون 2009) ؛ (Wittig وSchägger 2009) .

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

مجمعات Multiprotein (البلدان المتوسطية الشريكة) تلعب دورا حاسما في إشارة الخلية ، حيث يمكن العثور على معظم البروتينات في مجمعات وظيفية أو تنظيمية مع غيرها من البروتينات (سالي ، Glaeser وآخرون 2003). وهكذا ، ودراسة شبكات التفاعل البروتين البروتين يتطلب توصيف مفصل للبلدان المتوسطية الشريكة للحصول على فهم متكامل وظيفة البروتين والتنظيم. لتحديد وتحليل ، لا بد من فصل البلدان المتوسطية الشريكة في ظل الظروف المحلية. في هذا الفيديو ، وصفنا تحليل البلدان المتوسطية الشريكة التي كتبها زرقاء الأصلي بولي أكريلاميد الكهربائي للهلام (BN - PAGE). BN - PAGE هو الاسلوب الذي يسمح بالفصل بين البلدان المتوسطية الشريكة في التشكل الأصلية مع دقة أعلى من التي تقدمها الترشيح جل أو السكروز تنبيذ فائق الكثافة ، وبالتالي فهو مفيد لتحديد حجم MPC ، وتكوينها ، والوفرة النسبية (وSchägger فون Jagow 1991) ؛ (Schägger ، كريمر وآخرون 1994). بهذه الطريقة ، يتم فصل البروتينات وفقا لحجمها وشكلها الهيدروديناميكية في مصفوفة بولي أكريلاميد. هنا ، علينا أن نظهر للتحليل البلدان المتوسطية الشريكة من مجموع lysates الخلوية ، مشيرا إلى أن غسيل الكلى lysate هو خطوة حاسمة لجعل BN ​​- PAGE تنطبق على هذه العينات البيولوجية. باستخدام مزيج من أول BN البعد والبعد الثاني SDS - PAGE ، وتبين لنا أنه يمكن البلدان المتوسطية الشريكة مفصولة PAGE - BN تقسيمها إلى مزيد من ناخبيهم الفردية SDS - PAGE. يتم تنفيذ التصور من مكونات السياسة النقدية عند انتهاء الجل بواسطة immunoblotting القياسية. كمثال للتحليل بواسطة PAGE MPC - BN ، اخترنا 19S جيدا اتسم حقيقية النواة ، 20S ، وproteasomes 26S.

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

في هذا الفيديو ، وصفنا توصيف المجمعات multiprotein (البلدان المتوسطية الشريكة) من الأزرق الأصلي بولي أكريلاميد الكهربائي للهلام (BN - PAGE). في البعد الأول ، يتم فصل lysates الخلوية مدال بواسطة PAGE - BN لتحديد البلدان المتوسطية الشريكة الفردية. في البعد الثاني SDS - PAGE ، وتنقسم البلدان المتوسطية الشريكة من الاهتمام لتحليل من قبل ناخبيهم immunoblotting.

زرقاء هلام بولي أكريلاميد الأصلية الكهربائي (BN - PAGE) للتحليل من المجمعات Multiprotein Lysates الخليوي

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

130.8K Views.  University of Freiburg. في هذا الفيديو ، وصفنا توصيف المجمعات multiprotein (البلدان المتوسطية الشريكة) من الأزرق الأصلي بولي أكريلاميد الكهربائي للهلام (BN - PAGE). في البعد الأول ، يتم فصل lysates الخلوية مدال بواسطة PAGE - BN لتحديد البلدان المتوسطية الشريكة الفردية. في البعد الثاني SDS - PAGE ، وتنقسم الب...

Video: 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

Okay.Hi, my name is Celeste Peterson and I'm a fellow at Harvard University studying microbial bio film communities. So I am profiling the bacterial communities by looking at their 60 N sequence and how this sequence changes over time in space. And to do that, I'm using DDGE.This is not a normal acry of my well, it needs to be kept at 60 degrees when it's run and the buffer needs to be pumped continuously. So we're gonna warm up the buffer for about an hour until it's at 60 degrees, and then we can run the towel. Now I'm gonna wash this plate with dilute uc solution, peeling around for impurities, andone, light that up.Anything like that can cause regularities. It is important to wipe off all the water. So when I assemble the gel, I'll put that chip on the outside of the gel.So the gel will be in between these two pieces of glass and the chip will be on the outside and it won't affect the gel in between. So now I'm gonna assemble the gel in these clamps and then I squeeze them together and tighten by turning to the right. As with any gel, it's important to make sure that the bottom is flush.To prevent making, we now turn the gel around and put it in here to get sealed with the gasket. So it's important when you do this that the gel, that the plates are actually lined up with the gasket here. So these knobs on the side then go in and you press down to type the seal.So this is a 30 mil syringe. And this part and this part come with the bio red kit and it just on like this. And that's just to get rid of the excess water in the tubes.So put the tubing onto the gel to load the gel. First you put an adapter into the tubing and then a needle look with that small adapter and put it right in the center of the gel just so it's fairly below the bottom glass. Then this wire adapter goes into the long tubing and it's going to connect to equal syringes containing the high and the low density solution.So these are pre-prepared solutions. The blue one, I've added a dye here that just indicates that this is the most denaturing solution and this is the least fitting train solution, the low density one. And they have everything in them.And now I'm gonna to make, gonna be Actually I twice as much the week. And once you've got the tempt, you have about five to seven minutes to fill the gel before the colon rises. And now we fill the syringe juice with the solution.So I'm gonna fill it with about 15 the solution. And then to get rid of any air bubbles, I'll turn it upside down and squeeze gently until that top air bubble is gone. Now this little screw here is gonna go into this groove.This adapter is gonna line up with this clamp here, and it's gonna get C clamped in very tightly. And then this tubing is gonna attach to the syringe. And the same thing is done on the other side with the high density matrix where it's time to deliver.And so I'm gonna be turning this wheel in a steady clockwise motion. And first the blue solution is gonna go out first because that's the the most in nature, and it's gonna form the bottom part of the gel. And then after a little while, the clear solution will start filling in the gel and the Gradient will be formed.One moment is the gently on an angle to And as soon as you finish pouring the gel to wash out your tubes immediately because they will polymerize Going, It's the group there. And then press down. So this little, And on the other side, I'll do the same thing.Okay.Hi, my name is Celeste Peterson and I'm a fellow at Harvard University studying microbial bio communities. So I am profiling the bacterial communities by looking at their 60 F sequence and how the sequence changes over time in space. And to do that, I'm using DDGE.So DGE is Dena gel electrophoresis. And basically the idea is that there's a gradient and the DNA, as it runs down the gradient gets de natured. And so different 16 species will stop at different points along the gradient.And so it ends up being a fingerprint of the different sexiness species in the community. So the goal of my experiment is to see who is in the community and how that's changing over time and space. And I can run the sexiness DDD gel and then I can cut out the bands and identify the species that are represented in each community.So one of the biggest issues when, when doing A DDG gel is to figure out the gradient that's the best. And so that is just something that needs to be played around with and figuring out for a particular community which type of gradient. So the one I'm using today is 35 to 55%urea.One of the big problems that can happen with this technique is that smears come up in in the leans. And I found for that, for whatever reason, one of the best things to do is to run the gels for a really long time and then the smears disappear. Well, today I'm just using it for general bacterial communities, but this can also be used for a specific family of bacteria to see how those change over time, and those tend to be very clean gels.You can also look at mutations in single genes with DDGE.

تغيير طبيعة الكهربائي جل التدرج (DGGE)

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.

الكشف عن تجميع اميلويد بواسطة تغيير طبيعة شبه الكهربائي جل منظفات - Agarose

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

Identification of protein complexes with quantitative proteomics in S. cerevisiae

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as important intracellular signalling molecules. However, unlike proteins, lipids are small hydrophobic molecules that traffic primarily by poorly described nonvesicular routes, which are hypothesized to occur at membrane contact sites (MCSs). MCSs are regions where the endoplasmic reticulum (ER) makes direct physical contact with a partnering organelle, e.g., plasma membrane (PM). The ER portion of ER-PM MCSs is enriched in lipid-synthesizing enzymes, suggesting that lipid synthesis is directed to these sites and implying that MCSs are important for lipid traffic. Yeast is an ideal model to study ER-PM MCSs because of their abundance, with over 1000 contacts per cell, and their conserved nature in all eukaryotes. Uncovering the proteins that constitute MCSs is critical to understanding how lipids traffic is accomplished in cells, and how they act as signaling molecules. We have found that an ER called Scs2p localize to ER-PM MCSs and is important for their formation. We are focused on uncovering the molecular partners of Scs2p. Identification of protein complexes traditionally relies on first resolving purified protein samples by gel electrophoresis, followed by in-gel digestion of protein bands and analysis of peptides by mass spectrometry. This often limits the study to a small subset of proteins. Also, protein complexes are exposed to denaturing or non-physiological conditions during the procedure. To circumvent these problems, we have implemented a large-scale quantitative proteomics technique to extract unbiased and quantified data. We use stable isotope labeling with amino acids in cell culture (SILAC) to incorporate staple isotope nuclei in proteins in an untagged control strain. Equal volumes of tagged culture and untagged, SILAC-labeled culture are mixed together and lysed by grinding in liquid nitrogen. We then carry out an affinity purification procedure to pull down protein complexes. Finally, we precipitate the protein sample, which is ready for analysis by high-performance liquid chromatography/ tandem mass spectrometry. Most importantly, proteins in the control strain are labeled by the heavy isotope and will produce a mass/ charge shift that can be quantified against the unlabeled proteins in the bait strain. Therefore, contaminants, or unspecific binding can be easily eliminated. By using this approach, we have identified several novel proteins that localize to ER-PM MCSs. Here we present a detailed description of our approach. 

Here we describe a new quantitative proteomics technique for identifying protein complexes in Saccharomyces cerevisiae. In this study, we have used the SILAC method together with affinity purification followed by tandem mass spectrometry to identify with high specificity the binding partners of an ER protein, Scs2p.

تحديد بروتين المجمعات مع كمية البروتينات في البيرة س.

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as ...

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.

تغيير طبيعة جل بولي أكريلاميد الكهربائي اليوريا (PAGE اليوريا)

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 
&#160;

A basic protocol for the separation of DNA fragments using agarose gel electrophoresis is described.

الاغاروز الكهربائي للهلام لفصل أجزاء الحمض النووي

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.

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.

نهج جديد لتحليل مقارن لMultiprotein مجمعات بناء على<sup&gt; 15</sup&gt; N الأيض وصفها والكمية الطيف الكتلي

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.

رسم الخرائط على أساس فؤاد للخصائص مطاطا جدران الخلايا: في الأنسجة، الخلوية، وقرارات التحت خلوية

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.

الميوسين الاغاروز جل الكهربائي: ويسترن التنشيف للبروتينات سكرية عالية الوزن الجزيئي


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.

تنقية مجمعات الأصلية للدراسة الهيكلية باستخدام طريقة العلامة جنبا الى جنب الانجذاب

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

زرقاء هلام بولي أكريلاميد الأصلية الكهربائي (BN - PAGE) للتحليل من المجمعات Multiprotein Lysates الخليوي

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