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この実験的な手順のための完全かつ詳細なテキストプロトコルは、分子生物学におけるカレントプロトコールで使用可能です。ゲルサンドイッチの組み立てのため、ゲルの装置のための詳細なステップバイステップの手順はBioRad社のウェブサイト3に見つけることができます。 必要な機器： ガラス板（内側と外側） 10cmのセル：10.1 × 7.3センチメートル（インナープレート）、10.1 × 8.2センチメートル（外板）、バイオラッド 20cmのセル：20 × 20センチ（内側のプレート）、20 × 22.3センチメートル（外側のプレート）、バイオラッド 0.5〜1.5ミリメートルゲルのコームとスペーサー ゲルキャスティングスタンド 蓋とケーブルを含むゲル装置 高電圧電源 ヒーティングブロックまたは水浴 血清学的ピペットとピペットエイド ピペットとピペットチップ ゲルの乾燥機やスキャナ試薬およびソリューション： 尿素（超高純度） 40％アクリルアミド溶液（29:1） 10 × TBE溶液（トリス - ホウ酸、EDTAバッファー） 脱イオン、蒸留水 TEMED 30％（w / v）の過硫酸アンモニウム溶液 0.5 × TBEのソリューション ホルムアミド EDTA キシレンシアノール ブロモフェノールブルー メタノール エタノール<table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td>ボリューム</td><td colspan="3" style="text-align:center;"> 50ミリリットル </td><td colspan="3" style="text-align:center;"> 60ミリリットル </td><td colspan="3" style="text-align:center;"> 10ミリリットル </td></tr><tr><td>アクリルアミド濃度</td><td> 10パーセント </td><td> 12.5パーセント </td><td> 15パーセント </td><td> 10パーセント </td><td> 12.5パーセント </td><td> 15パーセント </td><td> 10パーセント </td><td> 12.5パーセント </td><td> 15パーセント </td></tr><tr><td>グラム尿素</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>ミリリットル40パーセントアクリル（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">脱イオン、蒸留水でボリュームをいっぱいに</td></tr></tbody></table>表1：一本鎖オリゴヌクレオチドを​​解決するための様々なアクリルアミド濃度とボリュームに必要な試薬およびソリューションゲルサンドイッチアセンブリとゲルの準備<ol><li>メーカーの説明にしたがってゲルを組み立て、ゲルキャスティング室3にゲルを固定してください。 0.5〜1.5ミリメートル厚のスペーサーを使用してください。 </li><li>分子生物学における現在のプロトコールにしたがって適切なポリアクリルアミド溶液を調製するか、表1に示します。 20センチメートル× 22センチ× 1.5ミリメートル、ゲル溶液の60mlの変性アクリルアミドゲル用と10.1 × 8.2センチメートル× 1 mmのゲル5 mlのゲル溶液が十分であるために。大きなゲルは、予想される製品/バンドは数個の塩基の範囲内にある場合、その後長くゲルは、一塩基の違いでバンドを解決するために使用されます。期待されるバンドが10以上のヌクレオチドが異なる場合、小さいゲルは、フラグメントを解消するために適したものとなる。あなたの分離要件に合ったポリアクリルアミドの割合を選択して、より高いポリアクリルアミドの濃度は、低分子量のフラグメントを解決します。アクリルアミドの希望する量の超純尿素と混合して使用してください。 0.5〜1 × TBEの最終濃度を取得し、脱イオン、蒸留水でボリュームを埋めるためにゲルのミックスにTBEバッファーを追加します。電子レンジで20秒のためのソリューションを熱し、軽く混ぜる。ソリューションが手に暖機するまで大きいゲルのボリュームに対してこの手順を繰り返します。 </li><li>すぐに血清用ピペット及び2枚のガラス板との間の自動ピペットエイドを使用してゲルを注ぐ。気泡を導入することは避けてください。コー​​ムを挿入し、ゲルが30〜60分間重合させる。 </li></ol>電気泳動装置をセットアップし、ゲルをプレラン<ol><li>指示3を製造するためによると鋳造室や組立それからゲル装置にゲルをマウント解除します。 </li><li>ガラス板がサブになるとバッファ（0.5〜1 × TBE）を実行して下側のバッファ室のウィットを埋めるバッファを2〜3 cmを合併。バッファを実行するとゲルの上部に上部バッファー槽がいっぱいに。 </li><li>慎重にコームを抜き取るとヒントをロードするピペットとゲルを使用して、実行中のバッファをウェルの中にすすいでください。 </li><li>高電圧電源へのケーブルのゲルシステムとプラグの蓋を取り付けます。あなたのサンプルをロードする前に、ゲルを加熱するとゲルから、残りの尿素を除去するために少なくとも30分間ゲルをプレランする必要があります。最適温度は45から55の間でなければなりません℃の温度が60 ° Cのバンドがスメアまたはガラス板が割れることができる可能性があるため。を避けるプレラン（ゲルあたり15〜25 W）のための一定の電力を選択してください。 </li></ol>サンプルの準備<ol><li>その間にあなたのサンプルを準備する。したがって、あなたのサンプルに2 ×ゲルローディングミックスの適切な量を追加します。ローディングミックスは、通常、90％ホルムアミド、0.5％EDTA、0.1％キシレンシアノール、0.1％ブロモフェノールブルーが含まれています。 </li><li>サンプルがゲル上にロードする前に、サンプルが数分間70から90 ° Cの間で試料を加熱することによって熱が変性でなければなりません。 </li></ol>ゲルをロードして実行<ol><li>プレランが終了したら、蓋を取り外し、尿素が井戸に溶出したとして前に説明したように徹底的にポケットをすすいでください。 </li><li>ポケットの底部から注意深くサンプルを適用します。気泡を導入することは避けてください。ローディングバッファーには、実行中に同じ条件を維持するために空のポケットに適用する必要があります。 </li><li>蓋を組み立て、55のゲルの温度を維持するために一定の電力でゲルを実行° Cプレランに似て。色素フロントがゲルの下端に達するまでのマーカー色素の移行を観察。実行期間は、使用されるアクリルアミドの割合、バッファーとゲルの厚さのイオン強度に依存しています。ランは、2〜4時間の間に続くことができます。 ゲルの温度を確認してください。ゲルの温度計は、実行時に右の温度を監視することができます。 </li></ol>ゲルを処理<ol><li>色素フロントがゲルの終わりに達したときに下側のバッファ室からゲルを出す。チャンバーからゲルを外し、クランプを緩めます。スペーサーを離れて引いて、慎重にガラス板を解体。必要な場合は上限も含むゲル部分を切り取る。 </li><li>注意深くゲル固定液（TBEプラス5〜10％のメタノールとエタノールの同じ濃度）で皿にゲルを移す。 5〜10分間溶液中にゲルをそのままにしてバッファを二度変更。 </li><li>ゲルは真空ゲル乾燥機やゲルの直接スキャンを使用してさらに処理するための準備ができています。 </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>バンドの鋭さは、いくつかの要因によって異なります。シャープなバンドのためにロードされたサンプルの量は、すなわち理想的には1から5μlの間、可能な限り小さくする必要があります。しかし、最大15μlをまだ受諾可能な解像度を確実にするロードすることができます。シャープなバンドの少ないサンプル量の結果。 </li><li>バンドの質も、ゲルの厚さに依存しています。たとえば0.75 mmの薄いゲルは?...

denaturing urea polyacrylamide gel electrophoresis (urea page)

尿素PAGEまたは変性尿素ポリアクリルアミドゲル電気泳動では、二次的DNAまたはRNAの構造を変性し、分子量に基づいて、ポリアクリルアミドゲルマトリックス中のそれらの分離に使用される6月8日Mの尿素を、採用しています。 2から500塩基間のフラグメントは、単一のヌクレオチドのような小さな長さの違いが、この方法1を用いて分離することができます。サンプルの移行は、選択されたアクリルアミドの濃度に依存する。ポリアクリルアミドの割合は低分子量のフラグメントを解消します。ゲルの実行中に尿素や45〜55 ° Cの温度の組み合わせは、非構造化DNAまたはRNA分子を分離することができます。 一般的にはこの方法は、一本鎖DNAまたはRNAの断片を、酵素的切断反応から次のような合成または標識オリゴヌクレオチドまたは製品を分析したり、精製するために必要です。 このビデオの記事では、変性尿素ポリアクリルアミドゲルを準備して実行する方法を示します。技術的なヒントは、元のプロトコルの1,2に加えて、含まれています。

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

Denaturing Urea Polyacrylamide Gel Electrophoresis Urea PAGE

変性尿素ポリアクリルアミドゲル電気泳動は、一本鎖DNAを分離または500ヌクレオチドの上限に達した時点でRNAに使用されます。熱変性サンプルと非構造化一本鎖との組み合わせで尿素は、その分子量に応じてゲルマトリックス内に移行する。

変性尿素ポリアクリルアミドゲル電気泳動（尿素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 ...

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

Research

JoVE Journal

Biology

Denaturing Urea Polyacrylamide Gel Electrophoresis (Urea PAGE)

78.6K ビュー.  Nanyang Technological University, Singapore - NTU. 変性尿素ポリアクリルアミドゲル電気泳動は、一本鎖DNAを分離または500ヌクレオチドの上限に達した時点でRNAに使用されます。熱変性サンプルと非構造化一本鎖との組み合わせで尿素は、その分子量に応じてゲルマトリックス内に移行する。

ビデオ: Denaturing Urea Polyacrylamide Gel Electrophoresis Urea PAGE

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.

変性剤濃度勾配ゲル電気泳動（DGGE）

生物学

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.

セミ変性洗剤-アガロースゲル電気泳動によりアミロイド凝集のためにスクリーニング

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.

ポリアクリルアミドゲルでのタンパク質の迅速かつ高感度コロイドクマG - 250染色

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.

細胞溶解物からの多タンパク質複合体の解析のための青いネイティブポリアクリルアミドゲル電気泳動（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 
&#160;

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

DNA断片の分離のためのアガロースゲル電気泳動

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架橋ポリアクリルアミドハイドロゲルの調製

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.

ムチンアガロースゲル電気泳動：高分子量の糖タンパク質のためのウェスタンブロッティング


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.

ブルー ネイティブ ポリアクリルアミドゲル電気泳動法と免疫ブロットを使用して培養されたひと細胞のミトコンドリア呼吸鎖複合体の解析

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.

ポリアクリルアミドゲル電気泳動と銀染色によるグリコサミノグリカンの検出

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.

ショウジョウバエの蛹の解剖学的、固定、視覚化

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