作者感谢用友科学为生命奖霍华德·休斯医学研究所的啊

1。准备磁带<ol><li>清洁的正面和背面板使用Invitrogen公司Nupage NOVEX微小胶体录像带，或其他商业微小胶体录像带。 </li><li>准备约1-3毫升的塑料环氧树脂，并根据制造商的指示，拌匀。 </li><li>适用于环氧树脂内缘的前面板上，两个板块接触时，磁带装配。 </li><li>通过背面板的底部缝滑入了一块过滤纸或纸板，装配时的录像带，以防止多余的环氧密封开幕。取出滤纸，只要这两个板块组装。 </li><li>将沿着一个密封的边缘粘结剂剪辑，并让磁带通宵坐在环氧树脂完全固化。 </li><li>电工胶带背面板的底部密封的缝隙。 </li></ol> 2。浇注胶<ol><li>侧臂瓶125毫升，加入以下内容:我总体积为9毫升试剂级水（表意文字- H 2 O）的2.25毫升4X Tris碱分离胶缓冲液pH值8.70，30.8％，丙烯酰胺/双丙烯酰胺3.50毫升，3.25毫升类型。 </li><li>德气体烧瓶的侧臂连接到一个真空装置，并放置在一个倒置的烧瓶上方塞10分钟的解决方案。 </li><li>转移去瓦斯的解决方案，以50毫升的一次性聚丙烯离心管9毫升，并添加过硫酸铵（APS）的10％ammounium 30μL和6μL氮，氮，N"，N-四甲基乙二胺（TEMED）和混合纷飞的解决方案，以避免引入气泡。 </li><li>立即倒入磁带的解决方案，允许它运行下来的录像带一侧，以防止形成凝胶板之间的任何气泡。录像带，以填补前（短）板的顶部边缘约2厘米。 </li><li>轻轻地添加0.5毫升-1 - 丁醇饱和1X解决凝胶缓冲，让凝胶聚合​​1小时。 </li></ol> 3。浇注浓缩胶<ol><li>加入125毫升侧臂瓶:4X Tris碱浓缩胶缓冲液pH值6.80，0.40毫升30.8％丙烯酰胺/双丙烯酰胺，表意文字-H 2 O 1.85毫升3毫升的总量为0.75毫升以下。 </li><li>德气体10分钟，如在第2步中所述的解决方案。 </li><li>而脱气浓缩胶溶液，倒入1 - 丁醇与1X解析解决凝胶顶部的凝胶缓冲饱和的。一旦与表意文字-H 2 O冲洗解决凝胶顶部倒掉水，并用吸水或滤纸滤纸吸收剩余的液体。 </li><li>取出3毫升的脱气的解决方案，并添加9μl和10％APS和2μLTEMED拌匀。 </li><li>很快倒在录像带上的解决方案，允许它运行录像带一侧，直到它附近的短前凝胶板的顶部边缘。 </li><li>清洁凝胶梳插入凝胶盒的顶部，磁带上的​​粘合剂剪辑放置在梳举行到位。让浓缩胶聚合为1小时至过夜。 </li></ol> 4。存储凝胶<ol><li>当凝胶完全聚合，除去粘结剂剪辑，并放置在一个塑料袋热封的录像带，或重新密封的塑料袋。 </li><li>倒入20毫升1X叠加凝胶缓冲区包含入袋0.1％的叠氮化钠。 </li><li>热封袋为5秒，并确保有袋无泄漏。 </li><li>储存在4°C凝胶</li></ol> 5。样品准备<ol><li>在薄壁离心管中，加入6.5μL蛋白质样品，为7.5μl2X NOVEX Tris-甘氨酸SDS样品缓冲液，1μL100毫米二硫苏糖醇（DTT）和表意文字-H 2 O（如有必要）的总体积15μL。 </li><li>由涡旋混合样品，并短暂离心​​管底部带来内容的样本。 </li> &lt;李&gt;变性的样品在70°C为10分钟。 </li></ol> 6。运行凝胶<ol><li>成立NOVEX凝胶仪器。 </li><li>加入Tris-甘氨酸电泳缓冲液200毫升，包含500μL的内宅Nupage抗氧化剂，和300毫升的Tris-甘氨酸缓冲的外室。 </li><li>加载到使用凝胶加载枪头井的变性蛋白质样品（15μL）。 </li><li>运行60-70分钟，200至五凝胶。 </li></ol> 7。染色的凝胶<ol><li>从凝胶仪器取出录像带，并打破由两个塑料凝胶板，除了用胶刀或硬油灰刀撬环氧树脂密封。仔细分离板胶，不撕凝胶​​照顾。 </li><li>冲洗与表意文字- H凝胶2轻轻摇动3倍。 </li><li>污渍轻轻摇动1小时Invitrogen公司SimplyBlue SafeStain为凝胶。 </li><li>倒出染色液和脱色，凝胶与表意文字-H 2 O </li></ol> 8。清洁磁带<ol><li>使用金​​属铲刮或关闭所有磁带环氧树脂剥离，使他们能够再次被重用。 </li><li>每个塑料凝胶板用温和的洗涤剂溶液洗，自来水蒸馏水冲洗。 </li></ol> 9。代表结果因为环氧树脂会形成水密封，（图1），录音带不会泄漏时，丙烯酰胺溶液泼到他们。此外，凝胶录音带可以轻易打开凝胶染色检索，环氧树脂，可去皮的磁带，让他们继续再利用。正如图2所示，凝胶蛋白标记和样品带​​，适用于常规蛋白电泳显示清晰的分离。 <img src="/files/ftp_upload/3465/3465fig1.jpg" alt="图1" /> 图1。 APPLI阳离子环氧树脂密封微小胶体录音带。一个薄薄的一层环氧树脂应用到磁带的边缘装配前。为了突出环氧树脂，它是假色红色在这个形象。 <img src="/files/ftp_upload/3465/3465fig2.jpg" alt="图2" /> 图2。 SDS的Tris-甘氨酸凝胶的蛋白质分离浇微小胶体磁带重新使用SDS-PAGE进行如上所述。以下电泳凝胶取出沾满SimplyBlue SafeStain为制造商的指示，从微小胶体录像带，并拍下使用传送白光。巷中号包含SeeBlue Plus2预染蛋白标准。在车道中号的蛋白质的分子量道尔顿（kDa）的凝胶右侧上表示。其他车道​​显示清除从表达了谷胱甘肽-S-转移酶（GST）标签蛋白的细菌裂解的蛋白质。所示的蛋白质洗净，从Glutathione珠GST标签蛋白洗脱之前。

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

没有利益冲突的声明。

蛋白质凝胶电泳是大多数分子生物学实验室不可或缺的方法。市售minigels提供了方便的较高水平，但可能是昂贵的。在这里，我们展示如何重新使用从的​​市售minigels的流行品牌的微小胶体录音带。此方法保留了方便，但有一​​个现成的凝胶源的市售minigels成本的一小部分。在此过程中的一个关键步骤是运用正确的金额环氧树脂完全密封录音带之前加入unpolymerized丙烯酰胺，以防止泄漏。只使用...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td> Invitrogen公司的Nupage的NOVEX微小胶体纸盒（1.0毫米） </td><td> Invitrogen公司</td><td> NC2010 </td><td>可在2，5，10，12，15和2D井</td></tr><tr><td>塑料环氧树脂</td><td>乐泰</td><td> 108771 </td><td></td></tr><tr><td>电工胶带</td><td>苏格兰威士忌</td><td></td><td></td></tr><tr><td> 125毫升侧臂瓶</td><td>耐热</td><td> 5360 </td><td></td></tr><tr><td> 4X Tris碱分离胶缓冲</td><td></td><td></td><td> 1.5 Tris碱的pH值8.7 </td></tr><tr><td>丙烯酰胺</td><td> Fisher Scientific则</td><td> 01065-500 </td><td></td></tr><tr><td> BIS-丙烯酰胺</td><td> MP Biomedicals， </td><td> 800173 </td><td></td></tr><tr><td> 30.8％的丙烯酰胺溶液</td><td></td><td></td><td> 30:0.8丙烯酰胺:双丙烯酰胺表意文字-H 2 O </td></tr><tr><td> 10％的APS </td><td> Fisher Scientific则</td><td> BP179-25 </td><td> 0.1克1毫升表意文字- H 2 O的过硫酸铵，立即使用前准备</td></tr><tr><td> TEMED </td><td> Arcos的有机物</td><td> 138450500 </td><td></td></tr><tr><td> 1 - 丁醇饱和1X分离胶缓冲</td><td></td><td></td><td> 5:2 1 - 丁醇1X分离胶缓冲</td></tr><tr><td> 4X Tris碱浓缩胶缓冲</td><td></td><td></td><td> 1.5 Tris碱pH值6.8 </td></tr><tr><td>凝胶梳</td><td> Invitrogen公司</td><td> NC3015 </td><td> 15好，1.0毫米厚度</td></tr><tr><td>脉冲封口机</td><td>趋势TEW电暖气设备</td><td></td><td></td></tr><tr><td>热封小包</td><td> kapak / Scotchpak </td><td></td><td> 1品脱大小（6.5"×8"） </td></tr><tr><td>凝胶存储缓冲区</td><td></td><td></td><td> 1X叠加0.1％叠氮化钠凝胶缓冲</td></tr><tr><td> NOVEX SDS样品缓冲液Tris-甘氨酸（2X） </td><td> Invitrogen公司</td><td> LC2676 </td><td></td></tr><tr><td>数码地面电视</td><td> FisherBiotech </td><td> BP172-5 </td><td></td></tr><tr><td> 10X的三Gylcine跑缓冲</td><td> Invitrogen公司</td><td> LC2675 </td><td> 10X股票1L:29.0Ğ三基地，144.0 g甘氨酸，10.0ĞSDS </td></tr><tr><td> Xcell的SureLock迷你细胞</td><td> Invitrogen公司</td><td> EI0001 </td><td></td></tr><tr><td>胶刀</td><td> Invitrogen公司</td><td> EI9010 </td><td></td></tr><tr><td> SimplyBlue安全染色</td><td> Invitrogen公司</td><td> LC6065 </tD&gt; <td></td></tr></tbody></table>

pouring and running a protein gel by reusing commercial cassettes

使用十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS - PAGE）分析蛋白质的评价是一种常见的技术，生物化学和分子生物学的研究人员1-4。执行每日蛋白质分析实验室，市售的聚丙烯酰胺凝胶（$ 10/gel）的成本可能是相当一段时间。为了降低成本，一些研究人员自己编写的聚丙烯酰胺凝胶。这些凝胶浇注的传统方法通常利用专门的设备和玻璃的凝胶板，可以是昂贵的，排除许多凝胶浇注和存储以供将来使用。此外，在清洗或浇注凝胶玻璃板处理可能会导致意外破损，创造一个安全隐患，这可能妨碍其使用在本科实验班。我们的协议演示如何同时倒入多个蛋白凝胶回收Invitrogen公司Nupage NOVEX微小胶体录音带，和廉价的马terials家装店购买。这种经济和精简的方法，包括了几个星期的方式储存在4°C凝胶。通过重新使用塑料胶盒从市售凝胶，实验室运行频繁的蛋白凝胶可显着节省成本，并帮助环境。此外，塑料胶录音带是极耐破损，这使得他们的本科生做实验教室的理想。

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

Pouring and Running a Protein Gel by reusing Commercial Cassettes

我们的协议演示如何倒在时间的多种蛋白质凝胶回收Invitrogen公司Nupage NOVEX微小胶体录音带，并在家装商店购买廉价的材料。这种经济和精简的方法，包括了几个星期的方式储存在4°C凝胶。通过重新使用塑料胶盒从市售凝胶，实验室运行频繁的蛋白凝胶可显着节省成本，并帮助环境。

浇注和运行的一种蛋白质凝胶重用商​​业磁带

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

pouring-and-running-a-protein-gel-by-reusing-commercial-cassettes

Research

JoVE Journal

Biology

24.8K 次观看.  University of Florida. 我们的协议演示如何倒在时间的多种蛋白质凝胶回收Invitrogen公司Nupage NOVEX微小胶体录音带，并在家装商店购买廉价的材料。这种经济和精简的方法，包括了几个星期的方式储存在4°C凝胶。通过重新使用塑料胶盒从市售凝胶，实验室运行频繁的蛋白凝胶可显着节省成本，并帮助环境。

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

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.

变性梯度凝胶电泳（DGGE）

科研

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.

半变性洗涤剂琼脂糖凝胶电泳筛选淀粉样蛋白聚合

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

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

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

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

无有机溶剂和醋酸，蛋白质与考马斯亮蓝G - 250凝胶染色

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

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

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

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

液相恢复GELFREE 8100分馏系统使用基于分子量分馏

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

Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

The dynamic nature of cellular machineries is frequently built on transient and/or weak protein associations. These low affinity interactions preclude stringent methods for the isolation and identification of protein networks around a protein of interest. The use of chemical crosslinkers allows the selective stabilization of labile interactions, thus bypassing biochemical limitations for purification. Here we present a protocol amenable for cells in culture that uses a homobifunctional crosslinker with a spacer arm of 12 &Aring;, dithiobis-(succinimidyl proprionate) (DSP). DSP is cleaved by reduction of a disulphide bond present in the molecule. Cross-linking combined with immunoaffinity chromatography of proteins of interest with magnetic beads allows the isolation of protein complexes that otherwise would not withstand purification. This protocol is compatible with regular western blot techniques and it can be scaled up for protein identification by mass spectrometry1.
Stephanie A. Zlatic and Pearl V. Ryder contributed equally to this work.

Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

The cell permeable crosslinker DSP [dithiobis-(succinimidyl propionate)] stabilizes transient and labile interactions in vivo, which allows their isolation using stringent protein complex purification techniques. Here we present a technique for crosslinking cells grown in culture followed by isolation of protein complexes by immunoprecipitation.

由不稳定的多蛋白质复合物的分离在体内控制细胞的交联和免疫磁性亲和层析

The dynamic nature of cellular machineries is frequently built on transient and/or weak protein associations. These low affinity interactions preclude ...

Protein Crystallization for X-ray Crystallography

Using the three-dimensional structure of biological macromolecules to infer how they function is one of the most important fields of modern biology. The availability of atomic resolution structures provides a deep and unique understanding of protein function, and helps to unravel the inner workings of the living cell. To date, 86% of the Protein Data Bank (rcsb-PDB) entries are macromolecular structures that were determined using X-ray crystallography.
To obtain crystals suitable for crystallographic studies, the macromolecule (e.g. protein, nucleic acid, protein-protein complex or protein-nucleic acid complex) must be purified to homogeneity, or as close as possible to homogeneity. The homogeneity of the preparation is a key factor in obtaining crystals that diffract to high resolution (Bergfors, 1999; McPherson, 1999).
Crystallization requires bringing the macromolecule to supersaturation. The sample should therefore be concentrated to the highest possible concentration without causing aggregation or precipitation of the macromolecule (usually 2-50 mg/ mL). Introducing the sample to precipitating agent can promote the nucleation of protein crystals in the solution, which can result in large three-dimensional crystals growing from the solution. There are two main techniques to obtain crystals: vapor diffusion and batch crystallization. In vapor diffusion, a drop containing a mixture of precipitant and protein solutions is sealed in a chamber with pure precipitant. Water vapor then diffuses out of the drop until the osmolarity of the drop and the precipitant are equal (Figure 1A). The dehydration of the drop causes a slow concentration of both protein and precipitant until equilibrium is achieved, ideally in the crystal nucleation zone of the phase diagram. The batch method relies on bringing the protein directly into the nucleation zone by mixing protein with the appropriate amount of precipitant (Figure 1B). This method is usually performed under a paraffin/mineral oil mixture to prevent the diffusion of water out of the drop.
Here we will demonstrate two kinds of experimental setup for vapor diffusion, hanging drop and sitting drop, in addition to batch crystallization under oil.

The 3-D structure of a molecule provides a unique understanding of how the molecule functions. The principal method for structure determination at near-atomic resolution is X-ray crystallography. Here, we demonstrate the current methods for obtaining three-dimensional crystals of any given macromolecule that are suitable for structure determination by X-ray crystallography.

X射线晶体学的蛋白质结晶

Using the three-dimensional structure of biological macromolecules to infer how they function is one of the most important fields of modern biology. The ...

Orthogonal Protein Purification Facilitated by a Small Bispecific Affinity Tag

Due to the high costs associated with purification of recombinant proteins the protocols need to be rationalized. For high-throughput efforts there is a demand for general methods that do not require target protein specific optimization1 . To achieve this, purification tags that genetically can be fused to the gene of interest are commonly used2 . The most widely used affinity handle is the hexa-histidine tag, which is suitable for purification under both native and denaturing conditions3 . The metabolic burden for producing the tag is low, but it does not provide as high specificity as competing affinity chromatography based strategies1,2.
Here, a bispecific purification tag with two different binding sites on a 46 amino acid, small protein domain has been developed. The albumin-binding domain is derived from Streptococcal protein G and has a strong inherent affinity to human serum albumin (HSA). Eleven surface-exposed amino acids, not involved in albumin-binding4 , were genetically randomized to produce a combinatorial library. The protein library with the novel randomly arranged binding surface (Figure 1) was expressed on phage particles to facilitate selection of binders by phage display technology. Through several rounds of biopanning against a dimeric Z-domain derived from Staphylococcal protein A5, a small, bispecific molecule with affinity for both HSA and the novel target was identified6 .
The novel protein domain, referred to as ABDz1, was evaluated as a purification tag for a selection of target proteins with different molecular weight, solubility and isoelectric point. Three target proteins were expressed in Escherishia coli with the novel tag fused to their N-termini and thereafter affinity purified. Initial purification on either a column with immobilized HSA or Z-domain resulted in relatively pure products. Two-step affinity purification with the bispecific tag resulted in substantial improvement of protein purity. Chromatographic media with the Z-domain immobilized, for example MabSelect SuRe, are readily available for purification of antibodies and HSA can easily be chemically coupled to media to provide the second matrix.
This method is especially advantageous when there is a high demand on purity of the recovered target protein. The bifunctionality of the tag allows two different chromatographic steps to be used while the metabolic burden on the expression host is limited due to the small size of the tag. It provides a competitive alternative to so called combinatorial tagging where multiple tags are used in combination1,7.

A novel and highly efficient two-step affinity chromatography protocol has been developed and is described in detail. The method is based on a small purification tag with two inherent affinities and is applicable to a wide range of target proteins with different properties.

由一个小型的双特异性亲和标签的推动正交蛋白质纯化

Due to the high costs associated with purification of recombinant proteins the protocols need to be rationalized. For high-throughput efforts there is a ...

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

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

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

提高蛋白质结晶的成功率随机Microseed矩阵筛选

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

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

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

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

蛋白质 - 蛋白质相互作用的双分子荧光互补的烟草原生质体和叶片可视化

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

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

在造血干细胞和祖细胞的成年小鼠颅骨骨髓体内四维跟踪

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

浇注和运行的一种蛋白质凝胶重用商​​业磁带

浇注和运行的一种蛋白质凝胶重用商业磁带