美国国立卫生研究院资助 R01 CA149976 支持这项工作。

所有程序必须按照机构动物使用和护理指南执行。该程序是为分析小鼠造血干细胞和祖细胞 (HSCs 和高压), 但可以适应其他细胞群体的分析而制定的。
1. 流式细胞仪分离小鼠 HSCs 和高压的研究
<ol><li>收获小鼠骨髓细胞如描述在文献6。 注: 在图 1和图 2所示的数据中, 骨髓是从 C57BL/6J 小鼠身上采集的。</li>
	<li>按照制造商的说明, 使用鼠标沿袭损耗套件执行骨髓细胞的血统耗尽。</li>
	<li>用抗体对细胞表面的标记进行孵化, 如7所述。在图 1和图 2所示的实验中, 使用了对 Sca-1、试剂盒、Flt3 和沿袭 (林) 标记 Mac1、Gr1、CD3e、B220 和 Ter119 的抗体。</li>
	<li>排序 2000-2万林 Sca1+Flt3 细胞 (HSCs ) 或林-Sca1 试剂盒+细胞(hp) 1.5 毫升离心管含有0.1 毫升磷酸盐缓冲盐水 (PBS) 与2% 胎牛血清。 注意: 对于图 1和图 2所示的数据, 细胞使用了一个带有70µM 喷嘴的流式分析仪进行排序。最大采集量为1.4 毫升。</li>
</ol>2. 样品准备
注意: 此步骤非常关键。仔细处理样品。清除上清液时, 要小心不要扰乱细胞颗粒。
<ol><li>离心机的1.5 毫升管包含排序的细胞在摆动斗转子在4°c, 500 x g, 5 分钟. 用吸管和100µL 吸管提示从细胞颗粒中取出所有但大约20µL。不要扰乱球团。<ol>
<li>如果样品包含少于5000个细胞, 并且被排序的样品的总容量少于0.2 毫升, 转移细胞首先到低捆绑0.2 毫升 PCR 管在离心之前。</li>
		<li>如果样品包含少于5000个细胞和容量是超过0.2 毫升, 用离心机在4°c, 500 x g, 为5分钟旋转细胞下来, 并且去除所有, 但200µL 的上清。再将剩余的200µL 中的颗粒细胞重新悬浮, 然后将细胞转移到0.2 毫升的 PCR 管中, 再次旋转。在第二次旋转后从颗粒中除去 20-30 µL, 然后重新悬浮细胞。</li>
	</ol>
</li>
	<li>并用重悬在试管中左上清的细胞。如有必要, 添加额外的 PBS 在2% 的血清/pbs (你也可以使用2% 牛血清白蛋白 (BSA)/pbs, 或 pbs 单独) 获得浓度 5 x104至 5 x 10 细胞/毫升。 注: 使用细胞计数法收集, 以估计用于悬浮细胞的体积。</li>
	<li>使用 2-5 µL 的重新悬浮细胞, 以确定准确的细胞浓度使用自动单元计数器 (按照制造商的指示) 或 hemocytometer9。</li>
	<li>将含有所需细胞数的重悬浮细胞转移到新的1.5 毫升管中。在图 1, 2000, 1000, 或 500 HSCs 或高压的实验被转移了。所需的细胞数取决于由西方印迹获得的信号的强度, 并根据经验确定。使用20µL 或100µL 离心吸管提示进行转移。</li>
	<li>离心机在摆动斗转子中的细胞在4°c, 500 x g, 5 分钟。</li>
	<li>根据制造商的指示, 生成100x 库存解决方案, 在亚砜中溶解蛋白酶体和磷酸酶抑制剂。</li>
	<li>通过稀释制造商提供的相当于蒸馏水的 4x Laemmli 样品缓冲器, 准备 2x Laemmli 样品缓冲器。添加适量的100x 股蛋白酶体和磷酸酶抑制剂, 以获得最终浓度2x 抑制剂在 2x Laemmli 样本缓冲区。 注: 2x Laemmli 样本缓冲区可以提前进行, 但蛋白酶体和磷酸酶抑制剂应立即添加, 然后添加 2x Laemmli 样本缓冲区 (加上抑制剂) 的细胞颗粒溶解细胞, 如以下步骤所述。</li>
	<li>小心地从细胞颗粒中取出部分清液, 并将其与试管中剩余的上清液结合在一起, 添加等量的 2x Laemmli 样品缓冲器加上蛋白酶体和磷酸酶抑制剂以达到500的最终浓度。单元格/µL 在 1x Laemmli 示例缓冲区中。 注意: 例如, 如果您在步骤2.5 中将2000个单元的总体积20µL 转移到一个管, 则在离心单元格 (步骤 2.6) 后, 应从颗粒中移除上清的18µL, 并将剩余的上清的2个µL 添加相等容积 (2 µL) 2x Laemmli 样品缓冲器, 以达到500细胞/µL 在 1x Laemmli 样品缓冲器中的最终浓度。</li>
	<li>并用重悬颗粒以尽快生成裂解液。在这一点上, 样品可以立即电泳通过 SDS 页凝胶, 或可在液体 N2冻结, 并存储在-80 °c, 以供将来使用。</li>
</ol>3. 电泳
<ol><li>将裂解物在95摄氏度或沸水中加热5分钟。</li>
	<li>Electrophorese 1-40 µL 的裂解物 (包含从500高达2万细胞当量) 通过 SDS 页凝胶在100V 使用标准协议10。 注: 在凝胶中装入的裂解液体积是由产生理想信号所需的细胞数决定的, 并取决于感兴趣的蛋白质的丰度和抗体的质量。图 1中的数据获得了2000、1000和500个细胞当量的裂解物。井的宽度应该在3毫米到1.5 毫米的范围内. 1.5 毫米的牙齿可以用剪刀从商业可用的梳子上剪下来。</li>
</ol>4. 转移和阻止
注: 执行11标准协议后的西方污点。下面简要介绍了这些步骤:
<ol><li>预处理氟 (PVDF) 膜与甲醇十年代, 然后简要地洗涤膜与蒸馏水。</li>
	<li>按照转移器具制造商提供的说明书中的说明, 将蛋白质从 SDS 页凝胶转移到膜上。</li>
	<li>转移后, 2 毫升5% 牛血清白蛋白 (BSA) 在 PBST (PBS 与0.1% 补体) 一夜间在4摄氏度以下标准协议11。</li>
</ol>5. 抗体标记
<ol><li>使用供应商推荐的抗体稀释, 在4摄氏度的情况下, 用抗体在一夜床上孵育细胞膜。 注: 在材料表中, 我们对 HSCs 和高压的抗体以及相应的抗体稀释进行了验证。使用标准程序执行抗体标记8。</li>
</ol>6. 检测
<ol><li>清洗膜3次, 每 PBST 5 分钟室温下清洗。</li>
	<li>室温下用2毫升增强化学发光缓冲剂孵化膜1分钟。根据制造商的说明或显影胶片和胶片处理器, 使用成像系统检测信号。</li>
</ol>

<ol>
	<li>Krutzik, P. O., Clutter, M. R., Nolan, G. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coordinate+analysis+of+murine+immune+cell+surface+markers+and+intracellular+phosphoproteins+by+flow+cytometry.">Coordinate analysis of murine immune cell surface markers and intracellular phosphoproteins by flow cytometry.</a> J Immunol. 175 (4), 2357-2365 (2005).</li><li>Du, J., 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=Signaling+profiling+at+the+single-cell+level+identifies+a+distinct+signaling+signature+in+murine+hematopoietic+stem+cells.">Signaling profiling at the single-cell level identifies a distinct signaling signature in murine hematopoietic stem cells.</a> Stem Cells. 30 (7), 1447-1454 (2012).</li><li>Signer, R. A., Magee, J. A., Salic, A., Morrison, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Haematopoietic+stem+cells+require+a+highly+regulated+protein+synthesis+rate.">Haematopoietic stem cells require a highly regulated protein synthesis rate.</a> Nature. 509 (7498), 49-54 (2014).</li><li>Wang, J., 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=A+differentiation+checkpoint+limits+hematopoietic+stem+cell+self-renewal+in+response+to+DNA+damage.">A differentiation checkpoint limits hematopoietic stem cell self-renewal in response to DNA damage.</a> Cell. 148 (5), 1001-1014 (2012).</li><li>Hoshii, T., 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=mTORC1+is+essential+for+leukemia+propagation+but+not+stem+cell+self-renewal.">mTORC1 is essential for leukemia propagation but not stem cell self-renewal.</a> J Clin Invest. 122 (6), 2114-2129 (2012).</li><li>Signer, R. A., 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=The+rate+of+protein+synthesis+in+hematopoietic+stem+cells+is+limited+partly+by+4E-BPs.">The rate of protein synthesis in hematopoietic stem cells is limited partly by 4E-BPs.</a> Genes Dev. 30 (15), 1698-1703 (2016).</li><li>Cai, 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=Runx1+deficiency+decreases+ribosome+biogenesis+and+confers+stress+resistance+to+hematopoietic+stem+and+progenitor+cells.">Runx1 deficiency decreases ribosome biogenesis and confers stress resistance to hematopoietic stem and progenitor cells.</a> Cell Stem Cell. , (2015).</li><li>Loven, J., 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=Revisiting+global+gene+expression+analysis.">Revisiting global gene expression analysis.</a> Cell. 151 (3), 476-482 (2012).</li><li>JoVE Science Education Database. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+a+Hemoacytometer+to+Count+Cells.">Using a Hemoacytometer to Count Cells.</a> Basic Methods in Cellular and Molecular Biology. , (2017).</li><li>JoVE Science Education Database. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Separating+Protein+with+SDS-PAGE.">Separating Protein with SDS-PAGE.</a> Basic Methods in Cellular and Molecular Biology. , (2017).</li><li>JoVE Science Education Database. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Western+Blot.">The Western Blot.</a> Basic Methods in Cellular and Molecular Biology. , (2017).</li><li>Jackson, R. J., Hellen, C. U., Pestova, T. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mechanism+of+eukaryotic+translation+initiation+and+principles+of+its+regulation.">The mechanism of eukaryotic translation initiation and principles of its regulation.</a> Nat Rev Mol Cell Biol. 11 (2), 113-127 (2010).</li><li>Eaton, S. L., 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=A+guide+to+modern+quantitative+fluorescent+western+blotting+with+troubleshooting+strategies.">A guide to modern quantitative fluorescent western blotting with troubleshooting strategies.</a> J Vis Exp. (93), e52099 (2014).</li></ol>

作者没有利益冲突要申报。

西方印迹是一种常见的技术检测特定的蛋白质和激活信号通路的组织或细胞。通过对通常使用的程序进行小的调整, 我们能够例行地检测15种不同的蛋白质 (材料表), 在 1.5万 HSCs, 在某些情况下, 在 500 HSCs。该协议中最关键的步骤是: 1) 精确计数单元格, 2) 最小化管之间的传输数, 3) 株溶藻单元格直接与 Laemmli 样本缓冲区。当株溶藻的细胞颗粒与 Laemmli 样品缓冲, 我们发现, 而不是删除所有...

造血干细胞 (HSCs) 是自我更新的细胞, 可以引起所有的血血统。它们是骨髓中相对稀少的细胞, 使得生物化学分析变得困难。适合于分析稀有细胞的方法, 如流式细胞术, 对于定量测定细胞表面标记物和胞内蛋白的相对数量非常有用。然而, 细胞内蛋白的分析需要使用细胞通透程序来实现抗体的存取, 而不是所有细胞表面的表位都能存活这些程序1,2。此外, 在不同的蛋白质亚型或裂解产物之间鉴别的抗体通常不能用于流式细胞术, 因此调查人员仍然依靠西方的印迹进行某些类型的分析。
裂解物细胞的印迹分析是大多数实验室的常规程序。细胞可以在保存细胞表面分子表位的本地条件下纯化, 细胞裂解物随后可以被制备和分析。然而, 用西方印迹对稀有的原代细胞种群中的蛋白质进行分析, 可以要求 euthanizing 大量的动物获得足够的细胞。通过对几个步骤进行小的调整, 传统的西方印迹协议能够检测出相对较小数量的 HSCs (500-1.5万, 取决于感兴趣的蛋白质) 的蛋白质。这些调整包括准确计数细胞, 仔细处理细胞颗粒, 减少细胞间的转移, 以最小化细胞损失, 并株溶藻一个定义数量的细胞与集中加载缓冲包含蛋白酶体和磷酸酶抑制剂。许多发表的报告包括西部污点获得与2万或更多 HSCs3,4,5,6,7;这个简单的程序将减少在4到40倍之间产生等效数据所需的细胞和实验动物的数量。该协议旨在以每个单元为基础对结果进行规范化, 而不是内部控制。这使得检测蛋白质水平的全面降低, 如果数据被规范化到内部控制, 可以忽略。本文描述了在细胞基础上规范化的重要性, 用于分析基因表达数据8, 同样的原理也适用于用西方印迹来定量蛋白质。此优化的协议对于需要分析少量单元格的任何人都是有用的。

<table>
	<tbody>
		<tr>
			<td>sodium dodecyl sulfate (SDS)</td>
			<td>Fisher Scientific</td>
			<td>BP166-500</td>
			<td>&#160;SDS-PAGE</td>
		</tr>
		<tr>
			<td>TEMED</td>
			<td>Fisher Scientific</td>
			<td>BP150-20</td>
			<td>&#160;SDS-PAGE</td>
		</tr>
		<tr>
			<td>30% Acrylamidel Bis solution</td>
			<td>Bio-Rad</td>
			<td>1610158</td>
			<td>&#160;SDS-PAGE</td>
		</tr>
		<tr>
			<td>1.5M Tris-HCl pH8.8</td>
			<td>TEKNOVA</td>
			<td>T1588</td>
			<td>&#160;SDS-PAGE</td>
		</tr>
		<tr>
			<td>1.0M Tris-HCl pH6.8</td>
			<td>TEKNOVA</td>
			<td>T1068</td>
			<td>&#160;SDS-PAGE</td>
		</tr>
		<tr>
			<td>Ammonium Persulfate</td>
			<td>Fisher Scientific</td>
			<td>BP179-25</td>
			<td>&#160;SDS-PAGE</td>
		</tr>
		<tr>
			<td>mini-protean 3 comb</td>
			<td>Bio-Rad</td>
			<td>1653360</td>
			<td>&#160;SDS-PAGE</td>
		</tr>
		<tr>
			<td> 
			Mini-PROTEAN Tetra Cell</td>
			<td>Bio-Rad</td>
			<td>1658005EDU&#160;</td>
			<td>&#160;SDS-PAGE</td>
		</tr>
		<tr>
			<td>&#160;Transfer System</td>
			<td>Bio-Rad</td>
			<td>1704155EDU</td>
			<td>transfer</td>
		</tr>
		<tr>
			<td>PowerPac HC Power Supply</td>
			<td>Bio-Rad</td>
			<td>1645052EDU&#160;</td>
			<td>electrophoresis</td>
		</tr>
		<tr>
			<td>Imaging System</td>
			<td>Bio-Rad</td>
			<td>1708195&#160;</td>
			<td>signal detection</td>
		</tr>
		<tr>
			<td>4x Laemmli Sample Buffer</td>
			<td>Bio-Rad</td>
			<td>1610747EDU&#160;</td>
			<td>sample preparation</td>
		</tr>
		<tr>
			<td>10x Tris/Glycine/SDS Electrophoresis Buffer</td>
			<td>Bio-Rad</td>
			<td>1610732EDU&#160;</td>
			<td>electrophoresis</td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Bio-Rad</td>
			<td>1610710EDU</td>
			<td>sample preparation</td>
		</tr>
		<tr>
			<td>&#160;RTA Mini PVDF Transfer Kit, for 40 blots</td>
			<td>Bio-Rad</td>
			<td>1704272&#160;</td>
			<td>transfer</td>
		</tr>
		<tr>
			<td>Protease Inhibitor Cocktail</td>
			<td>Sigma</td>
			<td>11697498001</td>
			<td>sample preparation</td>
		</tr>
		<tr>
			<td>Phosphatase Inhibitor Cocktailrs</td>
			<td>Gold Biotechnology</td>
			<td>GB-450-1</td>
			<td>sample preparation</td>
		</tr>
		<tr>
			<td>EIF4G</td>
			<td>Cell signaling</td>
			<td>2469</td>
			<td>WB antibody, validated in 1000 cells 
			antibody concentration: 1:1000 
			reference(figure): Fig2</td>
		</tr>
		<tr>
			<td>p-Rps6</td>
			<td>Cell signaling</td>
			<td>4858</td>
			<td>WB antibody,validated in 1000 cells 
			antibody concentration: 1:1000 
			reference(figure): Fig2</td>
		</tr>
		<tr>
			<td>actin(HRP conjugated)</td>
			<td>abcam</td>
			<td>ab49900</td>
			<td>WB antibody,validated in 500 cells 
			antibody concentration: 1:5000 
			reference(figure): Fig1, Fig2</td>
		</tr>
		<tr>
			<td>LC-3</td>
			<td>Abcam</td>
			<td>ab48394</td>
			<td>WB antibody,validated in 15000 cells 
			antibody concentration: 1:1000 
			reference(figure): ref5 (Fig5)</td>
		</tr>
		<tr>
			<td>S6</td>
			<td>Cell Signaling</td>
			<td>2317</td>
			<td>WB antibody,validated in 15000 cells 
			antibody concentration: 1:1000 
			reference(figure): ref5 (Fig4)</td>
		</tr>
		<tr>
			<td>4EBP1</td>
			<td>Cell Signaling</td>
			<td>9644</td>
			<td>WB antibody, validated in 15000 cells 
			antibody concentration: 1:1000 
			reference(figure): ref5 (Fig4)</td>
		</tr>
		<tr>
			<td>p-4EBP1</td>
			<td>Cell Signaling</td>
			<td>2855</td>
			<td>WB antibody,validated in 15000 cells 
			antibody concentration: 1:1000 
			reference(figure): ref5 (Fig4)</td>
		</tr>
		<tr>
			<td>TSC2</td>
			<td>Cell Signaling</td>
			<td>4308</td>
			<td>WB antibody,validated in 15000 cells 
			antibody concentration: 1:1000 
			reference(figure): ref5 (Fig4)</td>
		</tr>
		<tr>
			<td>HSP90</td>
			<td>Cell Signaling</td>
			<td>4877</td>
			<td>WB antibody,validated in 15000 cells 
			antibody concentration: 1:1000 
			reference(figure): ref5 (Fig5)</td>
		</tr>
		<tr>
			<td>PTEN</td>
			<td>Cell Signaling</td>
			<td>9188</td>
			<td>WB antibody,validated in 15000 cells 
			antibody concentration: 1:1000 
			reference(figure): ref5 (FigS1)</td>
		</tr>
		<tr>
			<td>mTOR</td>
			<td>Cell Signaling</td>
			<td>2983</td>
			<td>WB antibody,validated in 15000 cells 
			antibody concentration: 1:1000 
			reference(figure): ref5 (FigS1)</td>
		</tr>
		<tr>
			<td>p-mTOR</td>
			<td>Cell Signaling</td>
			<td>5536</td>
			<td>WB antibody,validated in 15000 cells 
			antibody concentration: 1:1000 
			reference(figure): ref5 (FigS1)</td>
		</tr>
		<tr>
			<td>PP2AB</td>
			<td>Cell Signaling</td>
			<td>4953</td>
			<td>WB antibody,validated in 15000 cells 
			antibody concentration: 1:1000 
			reference(figure): ref5 (FigS1)</td>
		</tr>
		<tr>
			<td>p-AMPKa</td>
			<td>Cell Signaling</td>
			<td>2535</td>
			<td>WB antibody,validated in 15000 cells 
			antibody concentration: 1:1000 
			reference(figure): ref5 (FigS1)</td>
		</tr>
		<tr>
			<td>actin</td>
			<td>Santa Cruz</td>
			<td>sc-47778</td>
			<td>WB antibody,validated in 15000 cells 
			antibody concentration: 1:3000 
			reference(figure): ref5 (Fig4)</td>
		</tr>
		<tr>
			<td>lineage depletion kit (mouse)</td>
			<td>Miltenyi Biotec</td>
			<td>130-090-858</td>
			<td>hematopoietic stem and progenitor cells purification</td>
		</tr>
		<tr>
			<td>LS columns</td>
			<td>Miltenyi Biotec</td>
			<td>130-041-305</td>
			<td>hematopoietic stem and progenitor cells purification</td>
		</tr>
		<tr>
			<td>SCF</td>
			<td>PeproTech</td>
			<td>250-03</td>
			<td>phosphorylation stimulation</td>
		</tr>
		<tr>
			<td>APC-Cy7 c-kit antibody</td>
			<td>ThermoFisher</td>
			<td>A15423</td>
			<td>cell sorting</td>
		</tr>
		<tr>
			<td>anti-B220</td>
			<td>ThermoFisher</td>
			<td>48-0452-82</td>
			<td>cell sorting</td>
		</tr>
		<tr>
			<td>anti-CD3e</td>
			<td>ThermoFisher</td>
			<td>48-0031-82</td>
			<td>cell sorting</td>
		</tr>
		<tr>
			<td>anti-Mac1</td>
			<td>ThermoFisher</td>
			<td>48-0112-82</td>
			<td>cell sorting</td>
		</tr>
		<tr>
			<td>anti-Gr1</td>
			<td>ThermoFisher</td>
			<td>48-5931-82</td>
			<td>cell sorting</td>
		</tr>
		<tr>
			<td>anti-Ter119</td>
			<td>ThermoFisher</td>
			<td>48-5921-82</td>
			<td>cell sorting</td>
		</tr>
		<tr>
			<td>Spacer Plates with 1.0 mm Integrated Spacers</td>
			<td>Bio-Rad</td>
			<td>1653311</td>
			<td>&#160;SDS-PAGE</td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Research Products International</td>
			<td>A30075</td>
			<td>membrane blocking</td>
		</tr>
		<tr>
			<td>serum</td>
			<td>Atlanta Biologicals</td>
			<td>A12450</td>
			<td>medium supplement</td>
		</tr>
		<tr>
			<td>cell counter</td>
			<td>Invitrogen</td>
			<td>AMQAF1000</td>
			<td>cell counting</td>
		</tr>
		<tr>
			<td>hemocytometer</td>
			<td>ThermoFisher</td>
			<td>&#160;S17040</td>
			<td>cell counting</td>
		</tr>
		<tr>
			<td>flim</td>
			<td>Denville Scientific</td>
			<td>E3012</td>
			<td>signal detection</td>
		</tr>
		<tr>
			<td>medical film processor</td>
			<td>Konica</td>
			<td>SRC-101A</td>
			<td>signal detection</td>
		</tr>
		<tr>
			<td>Supersignal West Femto Maximum Sensitivity Substrate</td>
			<td>Therom Scientific</td>
			<td>34096</td>
			<td>signal detection</td>
		</tr>
		<tr>
			<td>Allegra X-15R Centrifuge (Rotor SX4750A)</td>
			<td>Beckman Coulter</td>
			<td>X-15R</td>
			<td>sample preparation</td>
		</tr>
		<tr>
			<td>BLUEstain protein ladder</td>
			<td>Gold Biotechnology</td>
			<td>P007-500</td>
			<td>electrophoresis</td>
		</tr>
		<tr>
			<td>cell sorter</td>
			<td>BD</td>
			<td>FACSAria II</td>
			<td>sample preparation</td>
		</tr>
		<tr>
			<td>Incublock</td>
			<td>Denville Scientific</td>
			<td>I0510</td>
			<td>electrophoresis</td>
		</tr>
	</tbody>
</table>

a western blotting protocol for small numbers of hematopoietic stem cells

造血干细胞 (HSCs) 是罕见的细胞, 与小鼠骨髓中仅含有2.5万表型的长期再生 HSCs。对 HSCs (500-1.5万细胞) 的小数目进行了优化, 并适合于对其进行分析。表型 HSCs 纯化, 准确计数, 直接裂解 Laemmli 样品缓冲。用月桂酸钠聚丙烯酰胺凝胶电泳 (SDS 页) 分析了含有等量细胞的裂解物, 并根据标准的西方印迹协议制备并处理了该印迹。使用此协议, 可以对 2000-5000 HSCs 进行例行分析, 在某些情况下, 数据可以从很少500个单元中获得, 而大多数出版物中报告的2万到4万个单元格。本议定书应普遍适用于其他造血细胞, 并能通过标准实验室程序对少量细胞进行常规分析。

500-2000 纯化 HSCs 和高压钠灯的代表性结果如图 1和图 2所示。图 1中的β肌动蛋白信号可以从从一只老鼠的骨髓中纯化出来的 500 HSCs 和高压钠灯中检测出来。请注意, 将裂解物装入1.5 毫米井, 产生了比加载到3.0 毫米井更强的500高压信号。图 2是 EIF4G 的西方印迹和 Rps6 (p-Rps6) 的磷酸化, ?...

Watch this Scientific Journal Video about A Western Blotting Protocol for Small Numbers of Hematopoietic Stem Cells at JoVE.com

A Western Blotting Protocol for Small Numbers of Hematopoietic Stem Cells

通过对500只造血干细胞或祖细胞进行分析, 优化了标准的西方印迹协议。优化包括仔细处理细胞样本, 限制管道之间的传输, 并直接株溶藻 Laemmli 样本缓冲区中的细胞。

一种用于小数目造血干细胞的西方印迹协议

Hematopoietic stem cells (HSCs) are rare cells, with the mouse bone marrow containing only ~25,000 phenotypic long term repopulating HSCs. A Western ...

a-western-blotting-protocol-for-small-numbers-hematopoietic-stem

Research

JoVE Journal

Biochemistry

10.3K 次观看.  Cincinnati Children's Hospital Medical Center. 通过对500只造血干细胞或祖细胞进行分析, 优化了标准的西方印迹协议。优化包括仔细处理细胞样本, 限制管道之间的传输, 并直接株溶藻 Laemmli 样本缓冲区中的细胞。

视频: A Western Blotting Protocol for Small Numbers of Hematopoietic Stem Cells

Method for Identifying Small Molecule Inhibitors of the Protein-protein Interaction Between HCN1 and TRIP8b

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are expressed ubiquitously throughout the brain, where they function to regulate the excitability of neurons. The subcellular distribution of these channels in pyramidal neurons of hippocampal area CA1 is regulated by tetratricopeptide repeat-containing Rab8b interacting protein (TRIP8b), an auxiliary subunit. Genetic knockout of HCN pore forming subunits or TRIP8b, both lead to an increase in antidepressant-like behavior, suggesting that limiting the function of HCN channels may be useful as a treatment for Major Depressive Disorder (MDD). Despite significant therapeutic interest, HCN channels are also expressed in the heart, where they regulate rhythmicity. To circumvent off-target issues associated with blocking cardiac HCN channels, our lab has recently proposed targeting the protein-protein interaction between HCN and TRIP8b in order to specifically disrupt HCN channel function in the brain. TRIP8b binds to HCN pore forming subunits at two distinct interaction sites, although here the focus is on the interaction between the tetratricopeptide repeat (TPR) domains of TRIP8b and the C terminal tail of HCN1. In this protocol, an expanded description of a method for purifying TRIP8b and executing a high throughput screen to identify small molecule inhibitors of the interaction between HCN and TRIP8b, is described. The method for high throughput screening utilizes a Fluorescence Polarization (FP) -based assay to monitor the binding of a large TRIP8b fragment to a fluorophore-tagged eleven amino acid peptide corresponding to the HCN1 C terminal tail. This method allows &#39;hit&#39; compounds to be identified based on the change in the polarization of emitted light. Validation assays are then performed to ensure that &#39;hit&#39; compounds are not artifactual.

The interaction between HCN channels and their auxiliary subunit has been identified as a therapeutic target in Major Depressive Disorder. Here, a fluorescence polarization-based method for identifying small molecule inhibitors of this protein-protein interaction, is presented.

方法确定HCN1和TRIP8b之间蛋白 - 蛋白相互作用的小分子抑制剂

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are expressed ubiquitously throughout the brain, where they function to regulate the ...

科研

生物化学

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating these studies, reincorporation of detergent-solubilized membrane proteins into liposomes is a stochastic process where protein topology is impossible to enforce. This paper offers an alternative method to these challenging techniques that utilizes a liposome-based scaffold. Protein solubility is enhanced by deletion of the transmembrane domain, and these amino acids are replaced with a tethering moiety, such as a His-tag. This tether interacts with an anchoring group (Ni2+ coordinated by nitrilotriacetic acid (NTA(Ni2+)) for His-tagged proteins), which enforces a uniform protein topology at the surface of the liposome. An example is presented wherein the interaction between Dynamin-related protein 1 (Drp1) with an integral membrane protein, Mitochondrial Fission Factor (Mff), was investigated using this scaffold liposome method. In this work, we have demonstrated the ability of Mff to efficiently recruit soluble Drp1 to the surface of liposomes, which stimulated its GTPase activity. Moreover, Drp1 was able to tubulate the Mff-decorated lipid template in the presence of specific lipids. This example demonstrates the effectiveness of scaffold liposomes using structural and functional assays and highlights the role of Mff in regulating Drp1 activity.

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

This paper describes a method for assessing the interactions and assemblies of integral membrane proteins in vitro with various partner factors in a lipid-proximal environment.

使用脚手架脂质体重构脂质近端蛋白质 - 蛋白质相互作用<em&gt;体外</em

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating ...

SorLA and CLC:CLF-1-dependent Downregulation of CNTFR&#945; as Demonstrated by Western Blotting, Inhibition of Lysosomal Enzymes, and Immunocytochemistry

The heterodimeric cytokine Cardiotrophin-like Cytokine:Cytokine-like Factor-1 (CLC:CLF-1) targets the glycosylphosphatidylinositol (GPI)-anchored CNTFR&#945; to form a trimeric complex that subsequently recruits glycoprotein 130/Leukemia Inhibitory Factor Receptor-&#946; (gp130/LIFR&#946;) for signaling. Both CLC and CNTFR&#945; are necessary for signaling but so far CLF-1 has only been known as a putative facilitator of CLC secretion. However, it has recently been shown that CLF-1 contains three binding sites: one for CLC; one for CNTFR&#945; (that may promote assembly of the trimeric complex); and one for the endocytic receptor sorLA. The latter site provides high affinity binding of CLF-1, CLC:CLF-1, as well as the trimeric (CLC:CLF-1:CNTFR&#945;) complex to sorLA, and in sorLA-expressing cells the soluble ligands CLF-1 and CLC:CLF-1 are rapidly taken up and internalized. In cells co-expressing CNTFR&#945; and sorLA, CNTFR&#945; first binds CLC:CLF-1 to form a membrane-associated trimeric complex, but it also connects to sorLA via the free sorLA-binding site in CLF-1. As a result, CNTFR&#945;, which has no capacity for endocytosis on its own, is tugged along and internalized by the sorLA-mediated endocytosis of CLC:CLF-1.
The present protocol describes the experimental procedures used to demonstrate i) the sorLA-mediated and CLC:CLF-1-dependent downregulation of surface-membrane CNTFR&#945; expression; ii) sorLA-mediated endocytosis and lysosomal targeting of CNTFR&#945;; and iii) the lowered cellular response to CLC:CLF-1-stimulation upon sorLA-mediated downregulation of CNTFR&#945;.

The present protocol describes how Western blotting and immunocytochemistry combined with inhibition of lysosomal enzymes can be used to demonstrate the downregulation of Ciliary Neurotrophic Factor Receptor-&#945; (CNTFR&#945;), which is conveyed by the interaction between Cytokine-like Factor-1 (CLF-1) and the endocytic receptor sorLA.

SorLA和CLC:这表现在免疫印迹，溶酶体酶的抑制，和免疫组化CNTFRα的CLF-1依赖性下调

The heterodimeric cytokine Cardiotrophin-like Cytokine:Cytokine-like Factor-1 (CLC:CLF-1) targets the glycosylphosphatidylinositol (GPI)-anchored ...

Dissipative Microgravimetry to Study the Binding Dynamics of the Phospholipid Binding Protein Annexin A2 to Solid-supported Lipid Bilayers Using a Quartz Resonator

The dissipative quartz crystal microbalance technique is a simple and label-free approach to measure simultaneously the mass uptake and viscoelastic properties of the absorbed/immobilized mass on sensor surfaces, allowing the measurements of the interaction of proteins with solid-supported surfaces, such as lipid bilayers, in real-time and with a high sensitivity. Annexins are a highly conserved group of phospholipid-binding proteins that interact reversibly with the negatively charged headgroups via the coordination of calcium ions. Here, we describe a protocol that was employed to quantitatively analyze the binding of annexin A2 (AnxA2) to planar lipid bilayers prepared on the surface of a quartz sensor. This protocol is optimized to obtain robust and reproducible data and includes a detailed step-by-step description. The method can be applied to other membrane-binding proteins and bilayer compositions.

Here, we present an experimental protocol that can be employed to determine the binding affinities and mode of interaction of label-free phospholipid-binding protein annexin A2 with immobilized solid-supported bilayers (SLB) by simultaneously measuring the mass uptake and the viscoelastic properties of the protein annexin A2.

用石英谐振器研究磷脂结合蛋白附件 a2 与固体支持的脂质层结合动力学的耗散微重法

The dissipative quartz crystal microbalance technique is a simple and label-free approach to measure simultaneously the mass uptake and viscoelastic ...

Robust Comparison of Protein Levels Across Tissues and Throughout Development Using Standardized Quantitative Western Blotting

Western blotting is a technique that is commonly used to detect and quantify protein expression. Over the years, this technique has led to many advances in both basic and clinical research. However, as with many similar experimental techniques, the outcome of Western blot analyses is easily influenced by choices made in the design and execution of the experiment. Specific housekeeping proteins have traditionally been used to normalize protein levels for quantification, however, these have a number of limitations and have therefore been increasingly criticized over the past few years. Here, we describe a detailed protocol that we have developed to allow us to undertake complex comparisons of protein expression variation across different tissues, mouse models (including disease models), and developmental timepoints. By using a fluorescent total protein stain and introducing the use of an internal loading standard, it is possible to overcome existing limitations in the number of samples that can be compared within experiments and systematically compare protein levels across a range of experimental conditions. This approach expands the use of traditional western blot techniques, thereby allowing researchers to better explore protein expression across different tissues and samples.

This method describes a robust and reproducible approach for the comparison of protein levels in different tissues and at different developmental timepoints using a standardized quantitative western blotting approach.

利用标准化定量西方印迹技术, 对跨组织和整个开发过程中的蛋白质水平进行可靠比较

Western blotting is a technique that is commonly used to detect and quantify protein expression. Over the years, this technique has led to many advances ...

A Semi-High-Throughput Adaptation of the NADH-Coupled ATPase Assay for Screening Small Molecule Inhibitors

ATPase enzymes utilize the free energy stored in adenosine triphosphate to catalyze a wide variety of endergonic biochemical processes in vivo that would not occur spontaneously. These proteins are crucial for essentially all aspects of cellular life, including metabolism, cell division, responses to environmental changes and movement. The protocol presented here describes a nicotinamide adenine dinucleotide (NADH)-coupled ATPase assay that has been adapted to semi-high throughput screening of small molecule ATPase inhibitors. The assay has been applied to cardiac and skeletal muscle myosin II's, two actin-based molecular motor ATPases, as a proof of principle. The hydrolysis of ATP is coupled to the oxidation of NADH by enzymatic reactions in the assay. First, the ADP generated by the ATPase is regenerated to ATP by pyruvate kinase (PK). PK catalyzes the transition of phosphoenolpyruvate (PEP) to pyruvate in parallel. Subsequently, pyruvate is reduced to lactate by lactate dehydrogenase (LDH), which catalyzes the oxidation of NADH in parallel. Thus, the decrease in ATP concentration is directly correlated to the decrease in NADH concentration, which is followed by change to the intrinsic fluorescence of NADH. As long as PEP is available in the reaction system, the ADP concentration remains very low, avoiding inhibition of the ATPase enzyme by its own product. Moreover, the ATP concentration remains nearly constant, yielding linear time courses. The fluorescence is monitored continuously, which allows for easy estimation of the quality of data and helps to filter out potential artifacts (e.g., arising from compound precipitation or thermal changes).

A nicotinamide adenine dinucleotide (NADH)-coupled ATPase assay has been adapted to semihigh throughput screening of small molecule myosin inhibitors. This kinetic assay is run in a 384-well microplate format with total reaction volumes of only 20 &#181;L per well. The platform should be applicable to virtually any ADP producing enzyme.

用于筛选小分子抑制剂的NADH耦合ATPase分析的半高通量自适应

ATPase enzymes utilize the free energy stored in adenosine triphosphate to catalyze a wide variety of endergonic biochemical processes in vivo that would ...

A Rapidly Incremented Tethered-Swimming Maximal Protocol for Cardiorespiratory Assessment of Swimmers

Incremental exercise testing is the standard means of assessing cardiorespiratory capacity of endurance athletes. While the maximal rate of oxygen consumption is typically used as the criterion measurement in this regard, two metabolic breakpoints that reflect changes in the dynamics of lactate production/consumption as the work rate is increased are perhaps more relevant for endurance athletes from a functional standpoint. Exercise economy, which represents the rate of oxygen consumption relative to performance of submaximal work, is also an important parameter to measure for endurance-athlete assessment. Ramp incremental tests comprising a gradual but rapid increase in work rate until the limit of exercise tolerance is reached are useful for determining these parameters. This type of test is typically performed on a cycle ergometer or treadmill because there is a need for precision with respect to work-rate incrementation. However, athletes should be tested while performing the mode of exercise required for their sport. Consequently, swimmers are typically assessed during free-swimming incremental tests where such precision is difficult to achieve. We have recently suggested that stationary swimming against a load that is progressively increased (incremental tethered swimming) can serve as a &#34;swim ergometer&#34; by allowing sufficient precision to accommodate a gradual but rapid loading pattern that reveals the aforementioned metabolic breakpoints and exercise economy. However, the degree to which the peak rate of oxygen consumption achieved during such a protocol approximates the maximal rate that is measured during free swimming remains to be determined. In the present article, we explain how this rapidly incremented tethered-swimming protocol can be employed to assess the cardiorespiratory capacity of a swimmer. Specifically, we explain how assessment of a short-distance competitive swimmer using this protocol revealed that his rate of oxygen uptake was 30.3 and 34.8 mL&#8729;min-1&#8729;kg-1BM at his gas-exchange threshold and respiratory compensation point, respectively.

As opposed to measurement during free swimming, which presents inherent challenges and limitations, determination of important parameters of cardiorespiratory function for swimmers can be made using a more feasible and easier to administer tethered-swimming rapidly incremented protocol with gas exchange and ventilatory data collection.

游泳运动员心肺评估的快速递增的绑带游泳最大协议

Incremental exercise testing is the standard means of assessing cardiorespiratory capacity of endurance athletes. While the maximal rate of oxygen ...

Stepwise Dosing Protocol for Increased Throughput in Label-Free Impedance-Based GPCR Assays

Label-free impedance-based assays are increasingly used to non-invasively study ligand-induced GPCR activation in cell culture experiments. The approach provides real-time cell monitoring with a device-dependent time resolution down to several tens of milliseconds and it is highly automated. However, when sample numbers get high (e.g., dose-response studies for various different ligands), the cost for the disposable electrode arrays as well as the available time resolution for sequential well-by-well recordings may become limiting. Therefore, we here present a serial agonist addition protocol which has the potential to significantly increase the output of label-free GPCR assays. Using the serial agonist addition protocol, a GPCR agonist is added sequentially in increasing concentrations to a single cell layer while continuously monitoring the sample&#39;s impedance (agonist mode). With this serial approach, it is now possible to establish a full dose-response curve for a GPCR agonist from just one single cell layer. The serial agonist addition protocol is applicable to different GPCR coupling types, Gq Gi/0 or Gs and it is compatible with recombinant and endogenous expression levels of the receptor under study. Receptor blocking by GPCR antagonists is assessable as well (antagonist mode).

This protocol demonstrates real-time recording of full dose-response relationships for agonist-induced GPCR activation from a single cell layer grown on a single microelectrode using label-free impedance measurements. The new dosing scheme significantly increases throughput without loss in time resolution.

基于无标签阻抗的 GPCR 检测中用于提高吞吐量的逐步加量协议

Label-free impedance-based assays are increasingly used to non-invasively study ligand-induced GPCR activation in cell culture experiments. The approach ...

A High-Throughput Enzyme-Coupled Activity Assay to Probe Small Molecule Interaction with the dNTPase SAMHD1

Sterile alpha motif and HD domain-containing protein 1 (SAMHD1) is a pivotal regulator of intracellular deoxynucleoside triphosphate (dNTP) pools, as this enzyme can hydrolyze dNTPs into their corresponding nucleosides and inorganic triphosphates. Due to its critical role in nucleotide metabolism, its association to several pathologies, and its role in therapy resistance, intense research is currently being carried out for a better understanding of both the regulation and cellular function of this enzyme. For this reason, development of simple and inexpensive high-throughput amenable methods to probe small molecule interaction with SAMHD1, such as allosteric regulators, substrates, or inhibitors, is vital. To this purpose, the enzyme-coupled malachite green assay is a simple and robust colorimetric assay that can be deployed in a 384-microwell plate format allowing the indirect measurement of SAMHD1 activity. As SAMHD1 releases the triphosphate group from nucleotide substrates, we can couple a pyrophosphatase activity to this reaction, thereby producing inorganic phosphate, which can be quantified by the malachite green reagent through the formation of a phosphomolybdate malachite green complex. Here, we show the application of this methodology to characterize known inhibitors of SAMHD1 and to decipher the mechanisms involved in SAMHD1 catalysis of non-canonical substrates and regulation by allosteric activators, exemplified by nucleoside-based anticancer drugs. Thus, the enzyme-coupled malachite green assay is a powerful tool to study SAMHD1, and furthermore, could also be utilized in the study of several enzymes which release phosphate species.

SAMHD1 is a deoxynucleoside triphosphate triphosphohydrolase with critical roles in human health and disease. Here we present a versatile enzyme-coupled SAMHD1 activity assay, deployed in a 384-well microplate format, that allows for the evaluation of small molecules and nucleotide analogues as SAMHD1 substrates, activators, and inhibitors.

一种高通量酶偶联活性测定法，用于探测小分子与 dNTP 酶 SAMHD1 的相互作用

Sterile alpha motif and HD domain-containing protein 1 (SAMHD1) is a pivotal regulator of intracellular deoxynucleoside triphosphate (dNTP) pools, as this ...

RIBO-seq in Bacteria: a Sample Collection and Library Preparation Protocol for NGS Sequencing

The ribosome profiling technique (RIBO-seq) is currently the most effective tool for studying the process of protein synthesis in vivo. The advantage of this method, in comparison to other approaches, is its ability to monitor translation by precisely mapping the position and number of ribosomes on a mRNA transcript.
In this article, we describe the consecutive stages of sample collection and preparation for RIBO-seq method in bacteria, highlighting the details relevant to the planning and execution of the experiment.
Since the RIBO-seq relies on intact ribosomes and related mRNAs, the key step is rapid inhibition of translation and adequate disintegration of cells. Thus, we suggest filtration and flash-freezing in liquid nitrogen for cell harvesting with an optional pretreatment with chloramphenicol to arrest translation in bacteria. For the disintegration, we propose grinding frozen cells with mortar and pestle in the presence of aluminum oxide to mechanically disrupt the cell wall. In this protocol, sucrose cushion or a sucrose gradient ultracentrifugation for monosome purification is not required. Instead, mRNA separation using polyacrylamide gel electrophoresis (PAGE) followed by the ribosomal footprint excision (28-30 nt band) is applied and provides satisfactory results. This largely simplifies the method as well as reduces the time and equipment requirements for the procedure. For library preparation, we recommend using the commercially available small RNA kit for Illumina sequencing from New England Biolabs, following manufacturer&#39;s guidelines with some degree of optimization.
The resulting cDNA libraries present appropriate quantity and quality required for next generation sequencing (NGS). Sequencing of the libraries prepared according to the described protocol results in 2 to 10 mln uniquely mapped reads per sample providing sufficient data for comprehensive bioinformatic analysis. The protocol we present is quick and relatively easy and can be performed with standard laboratory equipment.

Here we describe the stages of sample collection and preparation for RIBO-seq in bacteria. Sequencing of the libraries prepared according to these guidelines results in sufficient data for comprehensive bioinformatic analysis. The protocol we present is simple, uses standard laboratory equipment and takes seven days from lysis to obtaining libraries.

细菌中的RIBO-seq:NGS测序样本收集和库准备协议

The ribosome profiling technique (RIBO-seq) is currently the most effective tool for studying the process of protein synthesis in vivo. The advantage of ...