Name: the fastest western in town: a contemporary twist on the classic western blot analysis
Uploaded: 2014-02-05T18:00:00
Description: Watch this Scientific Journal Video about The Fastest Western in Town: A Contemporary Twist on the Classic Western Blot Analysis at JoVE.com

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?...

<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>

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。

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

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

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 ...