我们想麦克马洪实验室的所有成员感谢他们的帮助和支持。这项研究是由美国国立卫生研究院的/ NCI R01 CA176839-01（以毫米）和一个院校研究与学术职业发展奖（IRACDA到JS）资助。

1。从细胞培养全细胞裂解液的制备<ol><li>将细胞培养皿置于冰上，加入冷PBS与5毫米EDTA（ 如 PBS 5毫升5毫米EDTA/10厘米2板）。从使用细胞刮烹调除去粘附的细胞，然后将细胞悬浮液转移到冷的15毫升锥形管中。 </li><li>在4℃下沉淀的细胞悬浮液通过低速离心以1000rpm（230×g离心）5分钟放置锥形管在冰上，小心地吸出上清液，而不破坏颗粒。 </li><li>用1毫升的PBS中含有5mM EDTA洗涤沉淀和细胞悬浮液转移到一个冷1.5毫升离心管中。在13,000 rpm（13,226×g离心）快速自旋悬浮液，持续10秒，以沉淀细胞。小心取出上清液，而不破坏冰的颗粒和地方管。 </li><li>重悬在25-200微升的粒料（根据颗粒大小， 即对于3×10 6个细胞加〜150微升的RIPA缓冲液）中RIPA缓冲液（50mM的Tris，150mM的氯化钠，0.5mM的EDTA，10mM的氟化钠，0.1％SDS，0.5％脱氧胆酸钠，1％NP-40），其中包含新加入的蛋白酶和磷酸酶抑制剂在2倍终浓度。短暂涡旋振荡每个试管中孵育悬挂在冰上20-30分钟。注意：有几种裂解缓冲液，可用于提取的蛋白，其以溶解的蛋白质和它们的变性剂（ 如 SDS，曲拉通X-100，或NP-40）的强度的能力上不同。 RIPA缓冲液是用于制备全细胞裂解液和破坏蛋白质 - 蛋白质相互作用是有用的。 </li><li>要在14,000 rpm（15,339 XG）5分钟，在4°C。创建一个岗位核分数，离心裂解液将上清转移到一个新的管而不破坏冰核富集的颗粒和地方管。注：核富集的颗粒也可能从管分离而提取的上清液。为了避免收集核富集的颗粒，直接将枪头我NTO粘性核富集的颗粒，并绘制了吸管，以便处置。 </li><li>根据使用这样的蛋白质定量测定作为BCA或布拉德福德制造商的说明确定各样品的蛋白质浓度。 </li></ol> 2。样品制备和电泳<ol><li>按照以下所列出的图表制备每​​个样品。作为替代方案，β-巯基乙醇，也可用于以2.5％的终浓度。然而，还原剂应该被加载在凝胶之前加入到每个样品一小时。总量占移液变化，只有20微升每个制备的样品，每装好。 <table border="1"><tbody><tr><td>试剂</td><td>减少的样本</td></tr><tr><td>蛋白质样本</td><td> X微升</td></tr><tr><td> 4×LDS样品缓冲液</td><td> 6微升</td></tr><tr><td> 500纳米红色的DTTucing代理</td><td> 2.4微升</td></tr><tr><td>去离子水</td><td>高达15.6微升</td></tr><tr><td>总成交量</td><td> 24微升</td></tr></tbody></table></li><li>热样品，在70℃10分钟。当样品加热，准备千毫升1X MES或MOPS电泳缓冲液（50毫升20倍的MES或MOPS电泳缓冲液加950毫升去离子水）。 MOPS运行缓冲可解决中型蛋白14-200 kDa的之间，而MES电泳缓冲液可解决2-200 kDa的之间的较小分子量的蛋白质具有较低的pKa值，其允许在MES电泳缓冲液至运行速度比MOPS 4更快。因此，这两个缓冲区都对比上，导致在蛋白质条带4的分离差异浓缩胶中离子的迁移作用。预留并结合200毫升1X的MES或MOPS用500μl抗氧化的电泳缓冲液，将用于填补上缓冲液槽中最小的I-细胞电泳装置。 </li><li>一旦样品被加热完毕，快速旋转的样本置于冰上将之前。 </li><li>对于预制Bis-Tris凝胶放入微型电池单元，用于电泳的装配遵循制造商的说明进行操作。注意：确保每个凝胶被以这样的方式取向，每个凝胶的短（小）盒向内朝向缓冲区核心和凝胶是水平和对缓冲区核心紧紧地压，以避免从上缓冲液槽泄漏。 </li><li>补上缓冲液槽与200毫升的1X的MES或MOPS电泳缓冲液含有抗氧化剂和下缓冲液槽与大约600毫升的1X的MES或MOPS电泳缓冲液的微型电池单元组成。额外的200-300毫升运行缓冲液可以保存供以后使用。然而，这是不推荐来回收电泳运行过程中用作缓冲液的pH和质量可以被改变的缓冲区。 </li><li>加载20微升每种蛋白质的SAMP勒到每个孔中。如果切片膜成各种条状，以探测多种抗体（ 即超过2），强烈建议加载2-5μl，以每个凝胶的第一个和最后一个孔的预染蛋白质标准，因为这将作为切割导板，用于分离的膜的不同部分，而不会影响该蛋白质条带的成像。 </li><li>运行凝胶在200V恒定35分钟以110-125毫安/凝胶在开始和每个凝胶70-80毫安在年底预期的电流。电泳完成时，跟踪染料迁移到铂丝沿缓冲区核心。 </li></ol> 3。蛋白质转移使用IBLOT干印迹系统<ol><li>通过破坏盒的侧粘接完全分离的微型凝胶（这可能产生一个噼啪噪声）。弃短（小）板，小心地从较长（开槽）盘转移到凝胶用去离子水容器中，除去位于底部凝胶的厚足部分。注意：在极少数情况下，凝胶可以连接到短板，而不是时间越长，开槽板。在这种情况下，丢弃该时间越长，开槽板和除去位于底部凝胶的厚右脚部。然后，小心地从短板转移凝胶去离子水。 </li><li>根据制造商的说明装配在阳极和阴极传输栈。注意：这两种硝化纤维素或PVDF膜上的蛋白质提供高质量和高效率的传输，并与荧光检测8兼容。然而，聚偏氟乙烯膜确实具有较高的结合容量（240微克/厘米3）除硝化纤维8（200微克/厘米3）。 </li><li>小心地取出任何气泡或空气凝胶和膜，因为这之间的残留将防止在传输过程中的蛋白条带任何失真。注意：不要修剪膜或被传呃栈以满足您的凝胶大小，因为这将导致阳极和阴极栈之间的直接接触。 </li><li>在正确组装印迹干燥设备上的传输协议栈，选择适当的电压计划（ 即方案三：20 V为7分钟），并开始传输。注：蛋白质大于150 kDa的趋向迁移速度比较慢，可能需要8-10分钟更长的传输时间。另外，在20％乙醇中5-10分钟的凝胶温育之前也将有助于提高这些大蛋白的转印效率。一旦传输程序完成后，设备会自动切断电流。最好是立即取出膜并继续阻止程序，以确保更好的蛋白质检测，避免晒出膜。 </li></ol> 4。红外荧光蛋白检测<ol><li>块膜中最小的Odyssey封闭缓冲液（不加吐温20），1小时0.8毫升/厘米2（S）过夜，在4°C。膜（次），也可阻止与脱脂奶粉，然而，这可能会导致较高的背景和干扰蛋白质的检测。 </li><li>在漫游所需的浓度阻断缓冲液含0.1％Tween-20中过夜，在4℃下在初级抗体孵育膜（次）对于双色检测，其中两个不同的抗原上可视化的相同印迹，两个初级抗体必须来自不同宿主物种和同时加入到所述膜（次）。注意：孵育所述膜（次）初级抗体之前，将膜（s）可以被划分成不同部分，以探测多种抗体（ 即大于2）在相同的膜。 </li><li>对于在TBS 10分钟加0.1％Tween-20中轻轻摇动洗膜（次）3倍。 </li><li>孵育膜（次）与荧光标记的二抗的稀释度1:20,000用于在Odysse的800纳米通道中的700nm的信道和1:6,000-1:10,000Ý阻断缓冲液含0.1％Tween-20的30〜60分钟（时间超过1小时温育膜（次）可能会增加背景）。从光保护膜（S）。注意：二次抗体需要从同一宿主物种衍生的，因为每个初级抗体和标记有不同的荧光团。 </li><li>对于在TBS 10分钟加0.1％Tween-20中轻轻摇动洗膜（次）3倍。从光保护膜（S）。 </li><li>膜（次）可被干燥或存储在任何TBS或PBS无Tween-20中，在4℃，直至扫描和成像用红外成像系统。荧光信号将保持稳定数月，如果适当的保护从光。 </li></ol>

<ol>
	<li>Burnette, W. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Western+blotting:+electrophoretic+transfer+of+proteins+from+sodium+dodecyl+sulfate--polyacrylamide+gels+to+unmodified+nitrocellulose+and+radiographic+detection+with+antibody+and+radioiodinated+protein+A.">Western blotting: electrophoretic transfer of proteins from sodium dodecyl sulfate--polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A.</a> Anal. Biochem. 112, 195-203 (1981).</li><li>Renart, J., Reiser, J., Stark, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transfer+of+proteins+from+gels+to+diazobenzyloxymethyl-paper+and+detection+with+antisera:+a+method+for+studying+antibody+specificity+and+antigen+structure.">Transfer of proteins from gels to diazobenzyloxymethyl-paper and detection with antisera: a method for studying antibody specificity and antigen structure.</a> Proc. Natl. Acad. Sci. U.S.A. 76, 3116-3120 (1979).</li><li>Towbin, H., Staehelin, T., Gordon, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophoretic+transfer+of+proteins+from+polyacrylamide+gels+to+nitrocellulose+sheets:+procedure+and+some+applications.">Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.</a> Proc. Natl. Acad. Sci. U.S.A. 76, 4350-4354 (1979).</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> NuPAGE Technical Guide. Life Technologies Corporation. IM-1001. 60, (2010).</li><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>Kubo, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+incubation+of+solutions+of+proteins+containing+dodecyl+sulfate+on+the+cleavage+of+peptide+bonds+by+boiling.">Effect of incubation of solutions of proteins containing dodecyl sulfate on the cleavage of peptide bonds by boiling.</a> Anal. Biochem. 225, 351-353 (1995).</li><li>Moos, M., Nguyen, N. Y., Liu, T. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reproducible+high+yield+sequencing+of+proteins+electrophoretically+separated+and+transferred+to+an+inert+support.">Reproducible high yield sequencing of proteins electrophoretically separated and transferred to an inert support.</a> J. Biol. Chem. 263, 6005-6008 (1988).</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=iBlot+Dry+Blotting+System.">iBlot Dry Blotting System.</a> Life Technologies Corporation. 25-0911, 84 (2012).</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=Western+Blot+Analysis.">Western Blot Analysis.</a> LI-COR Biosciences. 988-12622, (2012).</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=Odyssey+Infrared+Imaging+System+Application+Software+Version+3.030.">Odyssey Infrared Imaging System Application Software Version 3.030.</a> LI-COR Biosciences. , (2007).</li><li>Hambaeus, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+Milk+Composition.">Human Milk Composition.</a> Clin. Nutr. 54, 219-236 (1984).</li><li>DenHollander, N., Befus, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loss+of+antigens+from+immunoblotting+membranes.">Loss of antigens from immunoblotting membranes.</a> J. Immunol. Methods. 122, 129-135 (1989).</li></ol>

作者什么都没有透露。

在根据本协议产生高质量，定量Western印迹的关键步骤如下：1）测定蛋白质浓度，2）样品的制备; 3）阻挡膜和4）的初级抗体的质量和口径。 测定总蛋白浓度的精度可对定量蛋白质条带，因为红外荧光检测的灵敏度呈将扩增的样品的蛋白浓度发现任何轻微的差异产生了重大影响。为了避免在确定蛋白质浓度，生成的标准曲线与线性回归线或R平方值越接近1越好（R 2≥0.99</sup...

<html>为了使用抗体检测1-3蛋白质创造一个更好的方法，1977年和1979年间首次开发蛋白质印迹分析技术。此程序利用蛋白质与用第二抗体可视化，并通过放射自显影，UV光或过氧化物酶反应产物3检测目标蛋白电泳转移到膜上从SDS-PAGE凝胶。因此，这些相同的基本原则仍然被广泛使用在今天的Western blot检测协议。然而，这种经典的免疫印迹技术确实存在许多缺点，如缓慢电泳运行时间，低分辨率和人工蛋白条带，易患蛋白质降解，和有限的灵敏度，以及糟糕的数据质量4。因此，该协议描述了标准的Western blot检测程序，会产生更准确的定性和定量数据显著的进步和改善。 “&gt;本的Laemmli系统，用于分离各种使用SDS-PAGE的蛋白质是蛋白质印迹5最广泛使用的凝胶体系。尽管如此Western印迹系统的普及，这种方法可能会导致带内失真，分辨率的损失，并杂散带4，这可能是由于在分离胶，减少二硫键由于凝胶的不同的氧化还原状态，并且天冬氨酰-脯氨酰裂解再氧化的高pH（9.5）的蛋白质的脱氨基和烷基化反应的结果肽键是由于加热该蛋白质在Laemmli缓冲液（pH为5.2）4,6的的Bis-Tris凝胶系统，该系统工作在中性pH值（7.0），在Lamemmli系统提供了显著益处，该系统提高了蛋白质的稳定性，最小化蛋白质修饰，蛋白质保持其减少的状态，可以防止在电泳过程天冬氨酰-脯氨酰裂解，并且更重要的是，电泳运行时间是35分钟，4,6,7，另外，吨他的Bis-Tris凝胶体系也产生更清晰的条带，更高的分辨率和分离，提高了灵敏度产生更可靠的数据4。 同的Bis-Tris凝胶体系相结合，IBLOT干印迹系统采用高场强和电流从凝胶显著降低蛋白质的传输时间内到7分钟8膜。这个传输系统是基于其生成更有效和可靠的蛋白8的转印干印迹法。对于IBLOT传输系统的功效，以传统的传输系统的详细比较请参考以下网站： <a href="http://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/Protein-Expression-and-Analysis/Western-Blotting/Western-Blot-Transfer/iBlot-Dry-Blotting-System/iBlot-Dry-Blotting-Comparison-to-Semi-Dry-and-Wet.html">http://www.invitrogen.com/site/us/en/home/Products-and-Services/Application</a>s/Protein-Expression-and-Analysis/Western-Blotting/Western-Blot-Transfer/iBlot-Dry-Blotting-System/iBlot-Dry-Blotting-Comparison-to-Semi-Dry-and-Wet.html .它利用阳极和阴极的堆叠，这是由一个包含适当的传输缓冲器，即作为离子储层8的凝胶基质。在转移过程中，电解水有助于防止氧的产生从铜阳极产生，而不会导致带内失真8更一致的蛋白转移。此外，该传输系统还通过减少电极8之间的距离增加了传输速度。 虽然化学发光是最常见，最传统的方法进行蛋白质检测印迹分析，双色红外荧光检测大大提高了灵敏度，质量和Western blot检测数据的准确性。这种检测方法有两种最佳波长，700 nm处采用红外激光激发和800纳米，以生成具有最大信号-噪声比和灵敏度最高9清晰的图像数据。因此，两个靶蛋白可以同时使用700 nm和800 nm的荧光检测信道的相同的膜显影。更重要的是，红外荧光的线性度和动态范围允许为强，弱蛋白条带9的精确定量分析。此外，该协议提供了巨大的优点和改进对经典的Western印迹技术通过降低该实验的电泳和转移时间而不影响这些过程的有效性和使用红外荧光蛋白的检测，以产生更大的定性和定量蛋白质印迹数据。

<table border="0" cellpadding="0" cellspacing="0" width="624">
	<colgroup>
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="19">
			<td height="19" style="height:19px;width:297px;">BCA Protein Assay Kit</td>
			<td style="width:204px;">Thermo Scientific/Pierce</td>
			<td style="width:123px;">23225</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">4x NuPAGE&#174; LDS Sample Buffer&#160;</td>
			<td>Invitrogen/Life Technologies</td>
			<td>NP0007</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">10x NuPAGE&#174; Sample Reducing Agent&#160;</td>
			<td>Invitrogen/Life Technologies</td>
			<td>NP0004</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">20x NuPAGE&#174; MES SDS Running Buffer</td>
			<td>Invitrogen/Life Technologies</td>
			<td>NP0002</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">20x NuPAGE&#174; MOPS SDS Running Buffer</td>
			<td>Invitrogen/Life Technologies</td>
			<td>NP0001</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">NuPAGE&#174; Antioxidant</td>
			<td>Invitrogen/Life Technologies</td>
			<td>NP0005</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:297px;">NuPAGE&#174; Novex&#174; 4-12% Bis-Tris Gel 1.5 mm, 10 well&#160;</td>
			<td>Invitrogen/Life Technologies</td>
			<td>NP0335BOX</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:297px;">NuPAGE&#174; Novex&#174; 4-12% Bis-Tris Gel 1.5 mm, 15 well</td>
			<td>Invitrogen/Life Technologies</td>
			<td>NP0336BOX</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">SeeBlue&#174; Plus2 Pre-Stained Standard&#160;</td>
			<td>Invitrogen/Life Technologies</td>
			<td>LC5925</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">XCell SureLock&#174; Mini-Cell&#160;</td>
			<td>Invitrogen/Life Technologies</td>
			<td>EI0001</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">iBlot&#174; Transfer Stack, PVDF Regular</td>
			<td>Invitrogen/Life Technologies</td>
			<td>IB4010-01</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">iBlot&#174;Transfer Stack, PVDF Mini</td>
			<td>Invitrogen/Life Technologies</td>
			<td>IB4010-02</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">iBlot&#174; Transfer Stack, Nitrocellulose Regular</td>
			<td>Invitrogen/Life Technologies</td>
			<td>IB3010-01</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">iBlot&#174;Transfer Stack, Nitrocellulose Mini</td>
			<td>Invitrogen/Life Technologies</td>
			<td>IB3010-02</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">iBlot&#174; Gel Transfer Device&#160;</td>
			<td>Invitrogen/Life Technologies</td>
			<td>IB1001</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">&#946;-Actin</td>
			<td>Sigma</td>
			<td>A2228</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Phospho-BIM (S69)</td>
			<td>BD Biosciences</td>
			<td>N/A</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Total BIM</td>
			<td>Epitomics</td>
			<td>1036-1</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Phospho-ERK1/2 (T202/Y204)</td>
			<td>Cell Signaling Technology</td>
			<td>4370</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Total ERK1/2</td>
			<td>Cell Signaling Technology</td>
			<td>9107</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Odyssey&#174; Blocking Buffer&#160;</td>
			<td>LI-COR Biosciences</td>
			<td>927-40000&#160;</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">IRDye 800CW Goat anti-Rabbit IgG</td>
			<td>LI-COR Biosciences</td>
			<td>926-32211&#160;</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">IRDye 680LT Goat anti-Mouse IgG&#160;</td>
			<td>LI-COR Biosciences</td>
			<td>926-68020&#160;</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Western Incubation Box, Medium&#160;</td>
			<td>LI-COR Biosciences</td>
			<td>929-97205&#160;</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Odyssey&#174; Classic Imaging System</td>
			<td>LI-COR Biosciences</td>
			<td>N/A</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Odyssey&#174; Application Software V3.0.30</td>
			<td>LI-COR Biosciences</td>
			<td>N/A</td>
		</tr>
	</tbody>
</table>

the fastest western in town: a contemporary twist on the classic western blot analysis

还在积极利用当今最初成立于20世纪70年代末，西方印迹技术。然而，这种传统的Western印迹的方法有几个缺点，其中包括低质量的分辨率，杂散乐队，敏感性下降，而可怜的蛋白质的完整性。最新进展已经大大提高了标准的Western blot检测协议的许多方面产生较高的定性和定量数据。本的Bis-Tris凝胶系统，可以替代传统的Laemmli系统，产生更好的蛋白质分离和分辨率，保持蛋白完整性，并降低电泳至35分钟的运行时间。此外，IBLOT干印迹系统，极大地提高了蛋白质转移到在7分钟，这是在对比的是，往往更低效的冗长的传输时间传统的蛋白转移方法在膜的功效和速度。在这些高度创新的修改，蛋白d组合etection使用红外荧光成像效果更高质量，更准确，相比化学发光标准Western印迹法一致的数据。这种技术可以同时检测在相同的膜上两种不同的抗原通过利用被可视化在不同的荧光通道双色近红外染料。此外，荧光成像的线性度和宽动态范围允许为强，弱蛋白条带的精确定量。因此，该协议描述了重要的改进，以经典的Western印迹方法，其中这些改进显著提高数据的质量，同时大大降低了该实验中的性能时间。

双色红外荧光在相同的膜具有增强的灵敏度和最高的信号噪声比，以产生清晰的印迹图像检测强和弱带。通过双色Western印迹检测显现在两个700 nm和800 nm的荧光通道产生的典型的结果列举在图1和图2。在图1中最上面的Western印迹显示了并行（覆盖）检测总的ERK1 / 2在800纳米通道中的裂解物中的来自人2倍连续稀释的700纳米通道（红色）（绿色）和β-肌动蛋白的衍?...

Watch this Scientific Journal Video about The Fastest Western in Town: A Contemporary Twist on the Classic Western Blot Analysis at JoVE.com

The Fastest Western in Town: A Contemporary Twist on the Classic Western Blot Analysis

该协议探索的最新进展进行免疫印迹分析。这些新颖的改进采用的Bis-Tris凝胶系统用35分钟电泳运行时，一个7分钟的干印迹转移系统和红外荧光蛋白的检测和成像，其产生更高的分辨率，质量，灵敏度和西数据的改进的准确度。

最快在西镇：对经典蛋白质印迹分析现代风味

The Western blot techniques that were originally established in the late 1970s are still actively utilized today. However, this traditional method of ...

the-fastest-western-town-contemporary-twist-on-classic-western-blot

Research

JoVE Journal

Biology

26.9K 次观看.  University of California, San Francisco. 该协议探索的最新进展进行免疫印迹分析。这些新颖的改进采用的Bis-Tris凝胶系统用35分钟电泳运行时，一个7分钟的干印迹转移系统和红外荧光蛋白的检测和成像，其产生更高的分辨率，质量，灵敏度和西数据的改进的准确度。 

视频: The Fastest Western in Town: A Contemporary Twist on the Classic Western Blot Analysis

Western Blotting Using the Invitrogen NuPage Novex Bis Tris MiniGels

Western Blotting (or immunoblotting) is a standard laboratory procedure allowing investigators to verify the expression of a protein, determine the relative amount of the protein present in different samples, and analyze the results of co-immunoprecipitation experiments. In this method, a target protein is detected with a specific primary antibody in a given sample of tissue homogenate or extract. Protein separation according to molecular weight is achieved using denaturing SDS-PAGE. After transfer to a membrane, the target protein is probed with a specific primary antibody and detected by chemiluminescence.
Since its first description, the western-blotting technique has undergone several improvements, including pre-cast gels and user-friendly equipment. In our laboratory, we have chosen to use the commercially available NuPAGE electrophoresis system from Invitrogen. It is an innovative neutral pH, discontinuous SDS-PAGE, pre-cast mini-gel system. This system presents several advantages over the traditional Laemmli technique including: i) a longer shelf life of the pre-cast gels ranging from 8 months to 1 year; ii) a broad separation range of molecular weights from 1 to 400 kDa depending of the type of gel used; and iii) greater versatility (range of acrylamide percentage, the type of gel, and the ionic composition of the running buffer).
The procedure described in this video article utilizes the Bis-Tris discontinuous buffer system with 4-12% Bis-Tris gradient gels and MES running buffer, as an illustration of how to perform a western-blot using the Invitrogen NuPAGE electrophoresis system. In our laboratory, we have obtained good and reproducible results for various biochemical applications using this western-blotting method.

This technical article describes a standard western-blotting procedure using the commercially available NuPAGE electrophoresis Mini-Gel system from Invitrogen.

免疫印迹使用Invitrogen公司NuPage NOVEX双三MiniGels

Western Blotting (or immunoblotting) is a standard laboratory procedure allowing investigators to verify the expression of a protein, determine the ...

科研

生物学

Surgical Induction of Endolymphatic Hydrops by Obliteration of the Endolymphatic Duct

Surgical induction of endolymphatic hydrops (ELH) in the guinea pig by obliteration and obstruction of the endolymphatic duct is a well-accepted animal model of the condition and an important correlate for human Meniere's disease. In 1965, Robert Kimura and Harold Schuknecht first described an intradural approach for obstruction of the endolymphatic duct (Kimura 1965). Although effective, this technique, which requires penetration of the brain's protective covering, incurred an undesirable level of morbidity and mortality in the animal subjects. Consequently, Andrews and Bohmer developed an extradural approach, which predictably produces fewer of the complications associated with central nervous system (CNS) penetration.(Andrews and Bohmer 1989) 
The extradural approach described here first requires a midline incision in the region of the occiput to expose the underlying muscular layer. We operate only on the right side. After appropriate retraction of the overlying tissue, a horizontal incision is made into the musculature of the right occiput to expose the right temporo-occipital suture line. The bone immediately inferio-lateral the suture line (Fig 1) is then drilled with an otologic drill until the sigmoid sinus becomes visible. Medial retraction of the sigmoid sinus reveals the operculum of the endolymphatic duct, which houses the endolymphatic sac. Drilling medial to the operculum into the area of the endolymphatic sac reveals the endolymphatic duct, which is then packed with bone wax to produce obstruction and ultimately ELH. 
In the following weeks, the animal will demonstrate the progressive, fluctuating hearing loss and histologic evidence of ELH.

This video shows how to surgically obstruct the guinea pig's endolymphatic duct to produce endolymphatic hydrops.

内淋巴管闭塞手术诱导的内淋巴积水

Surgical induction of endolymphatic hydrops (ELH) in the guinea pig by obliteration and obstruction of the endolymphatic duct is a well-accepted animal ...

Western Blotting: Sample Preparation to Detection

Western blotting is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract. It uses gel electrophoresis to separate native or denatured proteins by the length of the polypeptide (denaturing conditions) or by the 3-D structure of the protein (native/ non-denaturing conditions). The proteins are then transferred to a membrane (typically nitrocellulose or PVDF), where they are probed (detected) using antibodies specific to the target protein.

Western blotting is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract.

免疫印迹检测样品制备

Western blotting is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract. It uses gel ...

Analysis of Cell Cycle Position in Mammalian Cells

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of cell proliferation underlies the pathology of diseases like cancer. As such there is great need to be able to investigate cell proliferation and quantitate the proportion of cells in each phase of the cell cycle. It is also of vital importance to indistinguishably identify cells that are replicating their DNA within a larger population. Since a cell&prime;s decision to proliferate is made in the G1 phase immediately before initiating DNA synthesis and progressing through the rest of the cell cycle, detection of DNA synthesis at this stage allows for an unambiguous determination of the status of growth regulation in cell culture experiments.
DNA content in cells can be readily quantitated by flow cytometry of cells stained with propidium iodide, a fluorescent DNA intercalating dye. Similarly, active DNA synthesis can be quantitated by culturing cells in the presence of radioactive thymidine, harvesting the cells, and measuring the incorporation of radioactivity into an acid insoluble fraction. We have considerable expertise with cell cycle analysis and recommend a different approach. We Investigate cell proliferation using bromodeoxyuridine/fluorodeoxyuridine (abbreviated simply as BrdU) staining that detects the incorporation of these thymine analogs into recently synthesized DNA. Labeling and staining cells with BrdU, combined with total DNA staining by propidium iodide and analysis by flow cytometry1 offers the most accurate measure of cells in the various stages of the cell cycle. It is our preferred method because it combines the detection of active DNA synthesis, through antibody based staining of BrdU, with total DNA content from propidium iodide. This allows for the clear separation of cells in G1 from early S phase, or late S phase from G2/M. Furthermore, this approach can be utilized to investigate the effects of many different cell stimuli and pharmacologic agents on the regulation of progression through these different cell cycle phases.
In this report we describe methods for labeling and staining cultured cells, as well as their analysis by flow cytometry. We also include experimental examples of how this method can be used to measure the effects of growth inhibiting signals from cytokines such as TGF-&beta;1, and proliferative inhibitors such as the cyclin dependent kinase inhibitor, p27KIP1. We also include an alternate protocol that allows for the analysis of cell cycle position in a sub-population of cells within a larger culture5. In this case, we demonstrate how to detect a cell cycle arrest in cells transfected with the retinoblastoma gene even when greatly outnumbered by untransfected cells in the same culture. These examples illustrate the many ways that DNA staining and flow cytometry can be utilized and adapted to investigate fundamental questions of mammalian cell cycle control.

Determining the cell cycle position of a population of cells, or understanding how signals affect proliferation, can be readily measured by flow cytometry using this protocol. We report a simple experimental approach to staining cells and quantifying their position in the cell cycle.

在哺乳动物细胞中细胞周期位置分析

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of ...

Imaging and 3D Reconstruction of Cerebrovascular Structures in Embryonic Zebrafish

Zebrafish are a powerful tool to study developmental biology and pathology in vivo. The small size and relative transparency of zebrafish embryos make them particularly useful for the visual examination of processes such as heart and vascular development. In several recent studies transgenic zebrafish that express EGFP in vascular endothelial cells were used to image and analyze complex vascular networks in the brain and retina, using confocal microscopy. Descriptions are provided to prepare, treat and image zebrafish embryos that express enhanced green fluorescent protein (EGFP), and then generate comprehensive 3D renderings of the cerebrovascular system. Protocols include the treatment of embryos, confocal imaging, and fixation protocols that preserve EGFP fluorescence. Further, useful tips on obtaining high-quality images of cerebrovascular structures, such as removal the eye without damaging nearby neural tissue are provided. Potential pitfalls with confocal imaging are discussed, along with the steps necessary to generate 3D reconstructions from confocal image stacks using freely available open source software.

Imaging of cerebrovascular development in larval zebrafish is described. Techniques to facilitate 3D imaging and modify cerebrovascular development using chemical treatments are also provided.

成像和胚胎斑马鱼脑血管结构的三维重建

Zebrafish are a powerful tool to study developmental biology and pathology in vivo.  The small size and relative transparency of zebrafish embryos make ...

Differentiation of Newborn Mouse Skin Derived Stem Cells into Germ-like Cells In vitro

Studying germ cell formation and differentiation has traditionally been very difficult due to low cell numbers and their location deep within developing embryos. The availability of a "closed" in vitro based system could prove invaluable for our understanding of gametogenesis. The formation of oocyte-like cells (OLCs) from somatic stem cells, isolated from newborn mouse skin, has been demonstrated and can be visualized in this video protocol. The resulting OLCs express various markers consistent with oocytes such as Oct4 , Vasa , Bmp15, and Scp3. However, they remain unable to undergo maturation or fertilization due to a failure to complete meiosis. This protocol will provide a system that is useful for studying the early stage formation and differentiation of germ cells into more mature gametes. During early differentiation the number of cells expressing Oct4 (potential germ-like cells) reaches ~5%, however currently the formation of OLCs remains relatively inefficient. The protocol is relatively straight forward though special care should be taken to ensure the starting cell population is healthy and at an early passage.

Differentiation of Newborn Mouse Skin Derived Stem Cells into Germ-like Cells In vitro

In recent years the formation of germ cells using in vitro culture methods has been demonstrated using several different types of somatic stem cells. This article will visually present the differentiation of newborn mouse derived stem cells to early oocyte-like cells.

新生小鼠皮肤干细胞分化成生殖细胞<em在体外</em

Studying  germ cell formation and differentiation has traditionally been very difficult  due to low cell numbers and their location deep within developing ...

A Guide to Modern Quantitative Fluorescent Western Blotting with Troubleshooting Strategies

The late 1970s saw the first publicly reported use of the western blot, a technique for assessing the presence and relative abundance of specific proteins within complex biological samples. Since then, western blotting methodology has become a common component of the molecular biologists experimental repertoire. A cursory search of PubMed using the term &#8220;western blot&#8221; suggests that in excess of two hundred and twenty thousand published manuscripts have made use of this technique by the year 2014. Importantly, the last ten years have seen technical imaging advances coupled with the development of sensitive fluorescent labels which have improved sensitivity and yielded even greater ranges of linear detection. The result is a now truly Quantifiable Fluorescence based Western Blot (QFWB) that allows biologists to carry out comparative expression analysis with greater sensitivity and accuracy than ever before. Many &#8220;optimized&#8221; western blotting methodologies exist and are utilized in different laboratories. These often prove difficult to implement due to the requirement of subtle but undocumented procedural amendments. This protocol provides a comprehensive description of an established and robust QFWB method, complete with troubleshooting strategies.

The advancement of western blotting using fluorescence has allowed detection of subtle changes in protein expression enabling quantitative analyses. Here we describe a robust methodology for detection of a range of proteins across a variety of species and tissue types. A strategy to overcome common technical problems is also provided.

指南现代荧光定量免疫印迹与故障排除策略

The late 1970s saw the first publicly reported use of the western blot, a technique for assessing the presence and relative abundance of specific proteins ...

Floral-Dip Transformation of Flax (Linum usitatissimum) to Generate Transgenic Progenies with a High Transformation Rate

Agrobacterium-mediated plant transformation via floral-dip is a widely used technique in the field of plant transformation and has been reported to be successful for many plant species. However, flax (Linum usitatissimum) transformation by floral-dip has not been reported. The goal of this protocol is to establish that Agrobacterium and the floral-dip method can be used to generate transgenic flax. We show that this technique is simple, inexpensive, efficient, and more importantly, gives a higher transformation rate than the current available methods of flax transformation.
In summary, inflorescences of flax were dipped in a solution of Agrobacterium carrying a binary vector plasmid (T-DNA fragment plus the Linum Insertion Sequence, LIS-1) for 1 - 2 min. The plants were laid flat on their side for 24 hr. Then, plants were maintained under normal growth conditions until the next treatment. The process of dipping was repeated 2 - 3 times, with approximately 10 - 14 day intervals between dipping. The T1 seeds were collected and germinated on soil. After approximately two weeks, treated progenies were tested by direct PCR; 2 - 3 leaves were used per plant plus the appropriate T-DNA primers. Positive transformants were selected and grown to maturity. The transformation rate was unexpectedly high, with 50 - 60% of the seeds from treated plants being positive transformants. This is a higher transformation rate than those reported for Arabidopsis thaliana and other plant species, using floral-dip transformation. It is also the highest, which has been reported so far, for flax transformation using other methods for transformation.

Floral-Dip Transformation of Flax Linum usitatissimum to Generate Transgenic Progenies with a High Transformation Rate

Here, we present a protocol to transform flax using Agrobacterium-mediated plant transformation via floral-dip. This protocol is simple to perform and inexpensive, yet yields a higher transformation rate than the current available methods for flax transformation.

亚麻花香浸改造（<em&gt;亚麻</em&gt;）生成的转基因后代具有高转化率

Agrobacterium-mediated plant transformation via floral-dip is a widely used technique in the field of plant transformation and has been reported to be ...

Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro

Red/near-infrared light therapy (R/NIR-LT), delivered by laser or light emitting diode (LED), improves functional and morphological outcomes in a range of central nervous system injuries in vivo, possibly by reducing oxidative stress. However, effects of R/NIR-LT on oxidative stress have been shown to vary depending on wavelength or intensity of irradiation. Studies comparing treatment parameters are lacking, due to absence of commercially available devices that deliver multiple wavelengths or intensities, suitable for high through-put in vitro optimization studies. This protocol describes a technique for delivery of light at a range of wavelengths and intensities to optimize therapeutic doses required for a given injury model. We hypothesized that a method of delivering light, in which wavelength and intensity parameters could easily be altered, could facilitate determination of an optimal dose of R/NIR-LT for reducing reactive oxygen species (ROS) in vitro.
Non-coherent Xenon light was filtered through narrow-band interference filters to deliver varying wavelengths (center wavelengths of 440, 550, 670 and 810nm) and fluences (8.5 x 10-3 to 3.8 x 10-1 J/cm2) of light to cultured cells. Light output from the apparatus was calibrated to emit therapeutically relevant, equal quantal doses of light at each wavelength. Reactive species were detected in glutamate stressed cells treated with the light, using DCFH-DA and H2O2 sensitive fluorescent dyes.&#160;
We successfully delivered light at a range of physiologically and therapeutically relevant wavelengths and intensities, to cultured cells exposed to glutamate as a model of CNS injury. While the fluences of R/NIR-LT used in the current study did not exert an effect on ROS generated by the cultured cells, the method of light delivery is applicable to other systems including isolated mitochondria or more physiologically relevant organotypic slice culture models, and could be used to assess effects on a range of outcome measures of oxidative metabolism.

Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro

Non-coherent Xenon light was passed through narrow-band interference and neutral density filters to deliver light of varying wavelength and intensity to cultured cells. This protocol was used to assess the effects of red/near-infrared light therapy on production of reactive species in vitro: no effects were observed using the tested parameters.

对于波长和红/近红外光治疗对氧化应激强度的范围影响的评估方法<em&gt;在体外</em

Red/near-infrared light therapy (R/NIR-LT), delivered by laser or light emitting diode (LED), improves functional and morphological outcomes in a range of ...

Imaging the Intracellular Trafficking of APP with Photoactivatable GFP

Beta-amyloid (A&#946;) is the major constituent of senile plaques found in the brains of Alzheimer&#8217;s disease patients. A&#946; is derived from the sequential cleavage of Amyloid Precursor Protein (APP) by &#946; and &#947;-secretases. Despite the importance of A&#946; to AD pathology, the subcellular localization of these cleavages is not well established. Work in our laboratory and others implicate the endosomal/lysosomal system in APP processing after internalization from the cell surface. However, the intracellular trafficking of APP is relatively understudied.
While cell-surface proteins are amendable to many labeling techniques, there are no simple methods for following the trafficking of membrane proteins from the Golgi. To this end, we created APP constructs that were tagged with photo-activatable GFP (paGFP) at the C-terminus. After synthesis, paGFP has low basal fluorescence, but it can be stimulated with 413 nm light to produce a strong, stable green fluorescence. By using the Golgi marker Galactosyl transferase coupled to Cyan Fluorescent Protein (GalT-CFP) as a target, we are able to accurately photoactivate APP in the trans-Golgi network. Photo-activated APP-paGFP can then be followed as it traffics to downstream compartments identified with fluorescently tagged compartment marker proteins for the early endosome (Rab5), the late endosome (Rab9) and the lysosome (LAMP1). Furthermore, using inhibitors to APP processing including chloroquine or the &#947;-secretase inhibitor L685, 458, we are able to perform pulse-chase experiments to examine the processing of APP in single cells.
We find that a large fraction of APP moves rapidly to the lysosome without appearing at the cell surface, and is then cleared from the lysosome by secretase-like cleavages. This technique demonstrates the utility of paGFP for following the trafficking and processing of intracellular proteins from the Golgi to downstream compartments.

While the transport of cell surface proteins is relatively easily studied, visualizing the trafficking of intracellular proteins is much more difficult. Here, we use constructs incorporating photoactivatable GFP and demonstrate a method to accurately follow the amyloid precursor protein from the Golgi apparatus to down-stream compartments and follow its clearance.

成像APP的细胞内运输与光活化GFP

Beta-amyloid (A&#946;) is the major constituent of senile plaques found in the brains of Alzheimer&#8217;s disease patients. A&#946; is derived from the ...