We would like to thank Andrea Portbury and Davin Townley-Tilson for critical reading of the manuscript and the NIH (grant # R01HL061656) for funding support.

注：所有实验均批准的机构动物照顾及使用委员会在北卡罗莱纳大学教堂山分校。 1，消费税主动脉瓣<ol><li>使用经批准的安乐死技术感兴趣的萌芽阶段，安乐死怀孕小鼠幼崽的发展所需要的胚胎一天（五）。 </li><li>用70％乙醇消毒的怀孕雌性的腹部。抬起小腹向上并远离内脏器官的皮肤和肌肉，并解剖开女性的下腹腔内，以允许访问的子宫角。 </li><li>要检索的子宫角，保持宫颈钳，小心切尾的镊子。提起子宫角，切割任何结缔组织，保持号角到位，并通过切割角子宫交界处的输卵管完成删除。 </li><li>将解剖子宫角中含有培养皿山坳ð的0.1M Tris缓冲液（pH7.6）中，并根据需要冲洗。然后，切开子宫角开口长度明智揭露胚胎囊。 </li><li>揭露胚胎，剖开胚胎囊，在胎盘和胎儿的交界处剪断脐带的船只来释放胚胎。放置在第二个培养皿用冷的0.1M Tris缓冲液中的解剖胚胎。返回第一个培养皿与其余undissected胚胎至冰上直至准备进行下一次的胚胎。 </li><li>为了提高胚胎内胸壁访问，杀头的胚胎。如果基因分型是必要的，删除和保存额外的组织片在Eppendorf管置于冰上。 </li><li>将胚胎在它的后面，切断胸壁垂直沿左肋侧，靠近前肢和水平横膈膜以上的可视化的心脏。然后，打开胸壁暴露心脏。 </li><li>使用镊子举行沿大血管高，使用产钳提起心脏，减少血管下方。该N，上面剪钳释放的心脏。如果肺血管保持完整无缺，肺部可能与心脏取出，然后再继续应予删除。 </li><li>切割并去除肺动脉上述阀的电平;在本文所描述的胚胎阶段，肺动脉稍微不透明的，这有助于从阀地区的歧视。切略低于肺动脉瓣，避免了小梁心室肌，并收集在步骤1.10中描述，如果这个地区的利益也。这里和在下一步骤中，使用毛细作用收集在裂解缓冲液中之前绘制过量Tris缓冲远离感兴趣的区域。 </li><li>小心地取出主动脉远端的主动脉瓣，正上方的阀的水平;像肺动脉，主动脉保持略微不透明在这些胚胎阶段，协助在其去除。切略低于主动脉瓣，再次避免了小梁心肌，并传送阀用钳子向含有蛋白酶和磷酸酶抑制剂在冰上的Eppendorf管中加入2μl裂解缓冲液（在材料表中所述）。 </li><li>重复步骤1.6-1.10，直到阀门已切除所有的胚胎，收集在一个单独的Eppendorf管中各阀门。如果所有的胚胎具有相同的基因型，所有的阀可被收集在一个单一的Eppendorf管中。 </li><li>如果基因分型将被执行以确定哪些阀门汇集在一起​​，立即将样品在裂解缓冲液中于-80℃直至进一步使用。样品可以进行基因分型（步骤2.1.1）后汇集。 </li></ol> 2，蛋白提取<ol><li>在冰上融化并离心收集组织在13,100 xg离心1分钟以收集在裂解缓冲液中的组织在管的底部。 <ol><li>如果胚胎进行基因分型，用吸管轻轻收集和含游泳池裂解液阀地区的胚胎具有类似的基因型。为了确保一个强大的b并且，汇集来自E13.5胚胎3-4阀的区域，每个样品建议。 </li><li>如果所有的胚胎是相同的基因型和所有阀门区域收集一起在步骤1.10，裂解整个样品，如在步骤2.2中所描述。 </li></ol></li><li>检查所生成的合并的样品的体积并加入裂解缓冲液中，以40微升的总体积;然后，破坏和均匀化，使用5毫米的不锈钢珠在Eppendorf管中的样品。操作lyser为2-4分钟，在50赫兹，按照制造商的说明。旋管短暂地（〜13,100 xg离心1分钟），并转移样品至新管中，留下的不溶性碎片之后。 </li><li>准备和SDS-PAGE和转移的后续Western blot分析不同的样品，下面的协议，如伊斯拉米等[9]。印迹为上样对照蛋白为蛋白定量是必不可少的。 </li></ol>

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	<li>Wirrig, E. E., Hinton, R. B., Yutzey, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+expression+of+cartilage+and+bone-related+proteins+in+pediatric+and+adult+diseased+aortic+valves.">Differential expression of cartilage and bone-related proteins in pediatric and adult diseased aortic valves.</a> J Mol Cell Cardiol. 50, 561-569 (2011).</li><li>Chakraborty, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Twist1+promotes+heart+valve+cell+proliferation+and+extracellular+matrix+gene+expression+during+development+in+vivo+and+is+expressed+in+human+diseased+aortic+valves.">Twist1 promotes heart valve cell proliferation and extracellular matrix gene expression during development in vivo and is expressed in human diseased aortic valves.</a> Dev Biol. 347, 167-179 (2010).</li><li>Lee, M. P., Yutzey, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Twist1+directly+regulates+genes+that+promote+cell+proliferation+and+migration+in+developing+heart+valves.">Twist1 directly regulates genes that promote cell proliferation and migration in developing heart valves.</a> PLoS One. 6 (e29758), (2011).</li><li>Peacock, J. D., Huk, D. J., Ediriweera, H. N., Lincoln, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sox9+transcriptionally+represses+Spp1+to+prevent+matrix+mineralization+in+maturing+heart+valves+and+chondrocytes.">Sox9 transcriptionally represses Spp1 to prevent matrix mineralization in maturing heart valves and chondrocytes.</a> PLoS One. 6 (e26769), (2011).</li><li>Chakraborty, S., Cheek, J., Sakthivel, B., Aronow, B. J., Yutzey, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shared+gene+expression+profiles+in+developing+heart+valves+and+osteoblast+progenitor+cells.">Shared gene expression profiles in developing heart valves and osteoblast progenitor cells.</a> Physiol Genomics. 35, 75-85 (2008).</li><li>Vogel, C., Marcotte, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insights+into+the+regulation+of+protein+abundance+from+proteomic+and+transcriptomic+analyses.">Insights into the regulation of protein abundance from proteomic and transcriptomic analyses.</a> Nat Rev Genet. 13, 227-232 (2012).</li><li>Meng, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+functional+Toll-like+receptors+2+and+4+in+human+aortic+valve+interstitial+cells:+potential+roles+in+aortic+valve+inflammation+and+stenosis.">Expression of functional Toll-like receptors 2 and 4 in human aortic valve interstitial cells: potential roles in aortic valve inflammation and stenosis.</a> Am J Physiol Cell Physiol. 294, 29-35 (2008).</li><li>Dyer, L. A., Wu, Y., Moser, M., Patterson, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BMPER-induced+BMP+signaling+promotes+coronary+artery+remodeling.">BMPER-induced BMP signaling promotes coronary artery remodeling.</a> Developmental Biology. 386, 385-394 (2014).</li><li>Eslami, A., Lujan, J. <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:+sample+preparation+to+detection.">Western blotting: sample preparation to detection.</a> J Vis Exp. (44), (2010).</li><li>Garside, V. C., Chang, A. C., Karsan, A., Hoodless, P. 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The authors have nothing to disclose.

量化蛋白质水平在早期胚胎小鼠和鸡胚心脏瓣膜区域的能力提供了理解的关键细胞信号传递活动阀发展的额外工具。我们这里所描述的协议不从标准蛋白分离方法有很大的不同。然而，通过修改某些关键步骤，我们已经成功地从非常小的样品尺寸相加得到的磷酸化蛋白。为实现这一结果，下面的步骤是特别重要的。以确保得到的优质蛋白，关键的是要保持样品冷却，不论在冰上或用冰冷却的缓冲?...

能够识别并量化蛋白表达水平是一个标准技术，动物 - 和基于细胞的实验。然而，尽管一个长期的兴趣在早期胚胎心脏瓣膜的开发，在开发过程中评估蛋白表达在这个特定的组织目前仅限于免疫组化在两个小鸡和鼠标1,2。在大多数模型生物体（ 如小鸡和小鼠）的显影阀定量蛋白表达的难度的部分是阀，从而限制了蛋白质，可以得到的数量的小尺寸。因此，为了定量分析，研究人员通常依靠RNA提取和扩增用于随后定量PCR或微阵列分析2-5。然而，RNA和蛋白的表达水平是不完全相关6，所以着眼于RNA的表达不能提供一个严格的帐户中发生的任何给定的信号的许多改变在发展途径，在不同的时间。基于此限制，在目前可用的方法，该方法的目标是开发一种协议，用于可靠地获得足够量的蛋白质从显影小鼠胚胎心脏瓣膜的区域为所发生的各种信号通路是在重要的变化的定量分析这个组织的成熟。 胚胎瓣膜已普遍从老鼠RNA分离和后续基因表达分析2-5解剖。然而，这些研究已不限于使用基因表达作为一个读出信号通路的激活，它不允许检测翻译后蛋白修饰，其可能会影响下游信号的检测。利用RNA分离技术为出发点，我们开始与解剖感兴趣的区域。因为我们关心的是检测磷酸化蛋白质，都说明信号拍拍在主动脉瓣发展（E13.5-14.5）的一个特定时期hway活性，我们进行的所有解剖在无磷酸盐的Tris缓冲液和收集的阀门与磷酸酶和蛋白酶抑制剂的Tris-基的裂解缓冲液。在我们的具体情况下，仅收集主动脉瓣区域，但肺动脉瓣区域可以很容易地在相同的时间来获得。该阀的区域分别然后均质化并结合当前用于研究在成年心脏瓣膜7蛋白表达的样品缓冲液。通过使用小样品体积（ 例如 ，2微升），并从胚胎中具有相同的基因型汇集阀的区域，我们能够检测磷酸化和未磷酸化的蛋白质在13.5 8天胚胎。因为阀的区域可以被冻结并存储在裂解缓冲液中，胚胎可以在需要集中之前进行基因分型。 这种技术拓宽了一套工具，可用于评估细胞signalin在开发过程中克途径，并提供了定量的恭维免疫组化，专门为开发心脏瓣膜。这种技术应该是有益的，不仅要发展心脏病，也给所有谁与早期胚胎的工作有兴趣，包含有限的组织区域发育生物学家。

<table border="0" cellpadding="0" cellspacing="0" width="972">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Name of Reagent/ Equipment</td>
			<td style="width:125px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:512px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Timed-pregnant mouse</td>
			<td style="width:125px;"></td>
			<td style="width:119px;"></td>
			<td>To be dissected at the embryonic stage of interest</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Stereoscopic microscope</td>
			<td style="width:125px;">Nikon</td>
			<td style="width:119px;">SMZ645</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">0.1 M Tris, pH 7.6</td>
			<td style="width:125px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Microscissors</td>
			<td style="width:125px;">Fine Science Tools</td>
			<td style="width:119px;">15003-08</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fine forceps, #5</td>
			<td style="width:125px;">Fine Science Tools</td>
			<td style="width:119px;">11251-30</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Dissecting needle holders</td>
			<td style="width:125px;">Ted Pella Inc.</td>
			<td style="width:119px;">13560</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Dissecting needles</td>
			<td style="width:125px;">Ted Pella Inc.</td>
			<td style="width:119px;">13561-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Micropipette, 20 &#956;l, with tips</td>
			<td style="width:125px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Lysis buffer</td>
			<td style="width:125px;"></td>
			<td style="width:119px;"></td>
			<td>50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA, 1% Triton</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PhosSTOP</td>
			<td style="width:125px;">Roche</td>
			<td style="width:119px;">4906845001</td>
			<td>Add 1 tablet to 10 ml lysis buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">TissueLyser LT</td>
			<td style="width:125px;">Qiagen</td>
			<td style="width:119px;">85600</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Stainless steel beads</td>
			<td style="width:125px;">Qiagen</td>
			<td align="right">69989</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Microcentrifuge</td>
			<td style="width:125px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

protein isolation from the developing embryonic mouse heart valve region

Western blot analysis is a commonly employed technique for detecting and quantifying protein levels. However, for small tissue samples, this analysis method may not be sufficiently sensitive to detect a protein of interest. To overcome these difficulties, we examined protocols for obtaining protein from adult human cardiac valves and modified these protocols for the developing early embryonic mouse counterparts. In brief, the mouse embryonic aortic valve regions, including the aortic valve and surrounding aortic wall, are collected in the minimal possible volume of a Tris-based lysis buffer with protease inhibitors. If required based on the breeding strategy, embryos are genotyped prior to pooling four embryonic aortic valve regions for homogenization. After homogenization, an SDS-based sample buffer is used to denature the sample for running on an SDS-PAGE gel and subsequent western blot analysis. Although the protein concentration remains too low to quantify using spectrophotometric protein quantification assays and have sample remaining for subsequent analyses, this technique can be used to successfully detect and semi-quantify phosphorylated proteins via western blot from pooled samples of four embryonic day 13.5 mouse aortic valve regions, each of which yields approximately 1 &#956;g of protein. This technique will be of benefit for studying cell signaling pathway activation and protein expression levels during early embryonic mouse valve development.

采用这种制备技术，我们能够从E13.5胚胎检测磷酸化的Smad 1,5,8（pSmad）在单一主动脉瓣区域。 如图1A所示 ，从甚至一个单一的阀区域分离的蛋白是足以检测微弱pSmad带。信号强度成比例地增大到汇集阀的区域的数量。重要的是，pSmad /β-肌动蛋白的比值保持在不同的样本大小（ 图1C）几乎恒定。由于可用的蛋白质的水平低，同样的印迹无法剥离，并再次探测总Smad蛋...

Watch this Scientific Journal Video about Protein Isolation from the Developing Embryonic Mouse Heart Valve Region at JoVE.com

Protein Isolation from the Developing Embryonic Mouse Heart Valve Region

The analysis of protein expression in young embryonic mouse valves has been hampered by the limited tissue available. This manuscript provides a protocol for preparing protein from developing embryonic mouse valve regions for western blot analysis.

从发展胚胎小鼠心脏瓣膜区蛋白分离

Western blot analysis is a commonly employed technique for detecting and quantifying protein levels. However, for small tissue samples, this analysis ...

protein-isolation-from-developing-embryonic-mouse-heart-valve

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9.2K 次观看.  University of North Carolina at Chapel Hill. The analysis of protein expression in young embryonic mouse valves has been hampered by the limited tissue available. This manuscript provides a protocol for preparing protein from developing embryonic mouse valve regions for western blot analysis.