这项工作是由美国国立卫生研究院（DP1CA174426卡洛斯F.巴尔巴斯）和上海科技大学，上海，中国（以JL）的支持。使用PyMOL的生成分子图形。

1.克隆<ol><li>获取已亚克隆入PET-28的表达载体系统和可应要求提供（PET-2F-ZIF）丙氨酸取代的两个手指ZIF域。34 </li><li> PCR扩增EmGFP从质粒翡翠-的pBAD与引物的5&#39;XmaI-EmGFP（5&#39;-GGAAATTG CCCGGG ATGGTGAGCAAGGGCGAGGAGCTGTTCAC-3&#39;; XMA我粗体位点）和3&#39;SacI位-EmGFP（5&#39;-CGGATCT GAGCTC TTACTTGTACAGCTCGTCCATGCCGAG-3&#39; ;大胆囊我的网站）。 <ol><li>使用5纳克模板DNA，10微升10X聚合酶缓冲液，1单位（U）Taq DNA聚合酶，0.2mM的每种dNTP和0.2中与剩余的体积为100微升的溶液的每种引物μM由蒸馏/去离子水。使用的循环条件：95℃下5分钟;的95℃的30个循环持续30秒，55℃30秒和72℃进行1分钟; 72℃下进行10分钟。通过凝胶extracti纯化PCR产物上，并确定使用分光光度计测量绝对260×50微克/ ml的DNA浓度。 </li></ol></li><li>消化的pET-2F-ZIF并用限制性编码EmGFP插入酶XMA我和 SacI我在推荐的缓冲液3小时，在37℃用酶10单位每1微克的DNA。可视化的琼脂糖凝胶电泳使用荧光嵌入染料，如溴化乙锭的DNA。 </li><li>净化用凝胶提取试剂盒将消化的DNA，并确定由分光光度计测量绝对260×50微克/ ml的DNA浓度。 </li><li>结扎纯化EmGFP编码DNA成50-100纳克的pET-2F-ZIF使用T4 DNA连接酶的1单位在室温至少1小时。对于最好的结果，执行使用6的连接反应：1插入到载体的摩尔比。 </li><li>解冻50微升冰的任何化学感受XL-1蓝大肠杆菌细胞和10-20纳克连接PET-2F-ZIF-EmGFP的轻轻混匀。 </li><li>保持在冰上30分钟。热休克在42℃为90秒，该混合物回收细胞在2ml超级最优肉汤与分解代谢物阻抑（SOC），1小时，在37℃振荡。 </li><li>传播100微升恢复文化上的LB培养基（LB）琼脂平板上用50微克/毫升卡那霉素和孵化O / N在37℃。 </li><li>翌日，接种6毫升超级肉汤（SB）或含50微克/毫升卡那霉素与来自LB琼脂平板上并培养O / N一个菌落在37℃的LB培养物。 注意：在使用菌落PCR引物5&#39;XmaI-EmGFP和3&#39;SacI位-EmGFP可用于标识要Miniprep试剂之前阳性克隆。 </li><li>纯化的pET-2F-ZIF-EmGFP通过小量制备和使用T7启动子的DNA测序确认质粒（5&#39;-TAATACGACTCACTATAGGG-3&#39;）。 </li></ol> 2.表达和纯化<ol><li>解冻50微升化学感受BL21 E的大肠杆菌冰细胞和100纳克PL的PET-2F-ZIF-EmGFP轻轻混匀asmid。变换为在步骤1.7-1.8中描述。 </li><li>翌日，接种含有50微克/毫升卡那霉素具有单个菌落10毫升LB培养基和生长O / N在37℃振荡。 </li><li>第二天，稀释10毫升的O / N培养的成1升LB培养基中补充有50微克/ ml卡那霉素，0.2％葡萄糖和100微米的ZnCl 2。生长的培养，在37℃振荡的光学密度在0.8 600纳米（OD 600）和诱导蛋白表达有2mM异丙基β-D-1-硫代半乳糖苷（IPTG）。 6小时后的生长在37℃，收获细胞，离心，在5000×g离心10分钟，在4℃。 注意：感应条件是高度可变的并且依赖于蛋白质的稳定性被表达。监测OD 600每30分钟，直到OD达到600的0.8。 </li><li>重悬细胞沉淀在5ml裂解缓冲液（50mM的Tris-HCl，500mM的氯化钠，100μM的ZnCl 2，1米M二硫苏糖醇（DTT），1mM的MgCl 2的，1mM苯甲基磺酰氟（PMSF）和10mM咪唑，pH 8.0）中。裂解冰上的细胞超声具有以下设置：50％的动力输出，5秒开/ 10秒关闭间隔4分钟的处理时间。避免解决方案过热。 </li><li>离心细胞裂解物在25000×g离心30分钟，在4℃，将上清液转移到一个新的收集管。对于最好的结果，执行在4℃以下所有步骤。 </li><li>运行通过一列预填充用1ml平衡的Ni-NTA浆液的上清液。用20ml洗涤缓冲液（50mM的Tris-HCl，500mM的氯化钠，100μM的ZnCl 2，1mM的DTT，1mM的MgCl 2的和的30mM咪唑，pH 8.0）中洗涤树脂。 </li><li>洗脱用5ml洗脱缓冲液（50mM的Tris-HCl，500mM的氯化钠，100μM的ZnCl 2，1mM的DTT，1mM的MgCl 2的，和300 mM咪唑，pH8.0）中的蛋白质。 </li><li>缓冲液交换用缓冲存储器的洗脱的蛋白（50毫的Tris-HCl，500mM的氯化钠，100μM的ZnCl 2，1mM的DTT，1mM的MgCl 2的10％的甘油，pH 8.0）中，并使用根据制造商的说明书通过离心分离旋集中器集中的蛋白以至少40微米。 </li><li>用BCA或Bradford试验测定蛋白质浓度。拌2微升纯化的蛋白与2微升2×SDS-PAGE上样染料，煮沸，在95℃下进行10分钟，然后在4％解决-20％Tris-甘氨酸SDS-PAGE来评估蛋白质纯度（ 图2）。 </li></ol> 3.蛋白质存储<ol><li>分装浓缩的蛋白质，闪存冷冻在液氮中，并储存在-80℃。对于EmGFP融合蛋白，用铝箔覆盖的管中，以保护该蛋白质从光。 </li><li>避免反复冷冻和蛋白质溶液的解冻。注意：ZIF-融合蛋白是在这些条件下稳定至少1个月。不当或&gt;4个月存储可能会导致蛋白质沉淀或EmGFP的漂白。 </li></ol> 4.蛋白转导<ol><li>维持HeLa细胞中含有10％的Dulbecco氏改良的Eagle氏培养基（DMEM）（体积/体积）胎牛血清（FBS）和1％的抗生素-抗真菌剂在完全湿润气氛，用5％的CO 2在37℃。 注：细胞传代不推荐用于蛋白转导的30倍以上。 </li><li>预涂24孔板用500μl的多聚赖氨酸的50微克/毫升30至60分钟，在25℃下。种子细胞到24孔板，每孔2×10 5个细胞的密度。在接种后24小时，或一旦细胞是在80％-90％汇合，从各取出介质井和洗涤用500μl的预热无血清DMEM（SFM）。 </li><li>向每个孔中，加入含有2微米的ZIF-EmGFP蛋白和100μM的氯化锌加入250μlSFM的。孵育细胞，在37℃1小时。注：ZIF域进入细胞公关imarily通过巨吞饮，34是能量依赖性机制。因此，细胞必须在37℃下孵育高效蛋白质内化。 </li><li>从细胞用500μl钙和无镁的除去介质和洗涤三次Dulbecco氏磷酸盐缓冲盐水（DPBS），补充有肝素的0.5毫克/毫升。注意：肝素是需要除去可能复杂化下游分析表面结合的蛋白质。 </li><li> （可选）重复治疗最多三次以增强递送。 </li><li>肝素洗涤消化用0.05％胰蛋白酶-EDTA在37℃下2分钟后，立即分离处理的细胞。重悬在0.5ml DPBS中补充有1％FBS的分离的细胞。 </li><li>测量使用异硫氰酸荧光素（FITC）通道（ 图3），每个样品通过流式细胞仪35的荧光强度。调整前向散射（FSC）和侧向散射（SSC）放置的人口息上的规模，确保种群具有不同性质彼此解决。 </li><li>收集万实况事件对于每个样品，并分析利用流式细胞仪从与ZIF-EmGFP处理以那些只带有SFM处理的细胞的HeLa细胞的数据分析软件。36标准化荧光的数据。 </li></ol>

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basics+of+flow+cytometry.">Basics of flow cytometry.</a> Methods Mol. Biol. 91, 1-24 (1998).</li><li>Mercer, A. C., Gaj, T., Sirk, S. J., Lamb, B. M., Barbas, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+endogenous+human+gene+expression+by+ligand-inducible+TALE+transcription+factors.">Regulation of endogenous human gene expression by ligand-inducible TALE transcription factors.</a> ACS Synth. Biol. 3, 723-730 (2014).</li><li>Segal, D. J., Crotty, J. W., Bhakta, M. S., Barbas, C. F., Horton, N. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+Aart,+a+designed+six-finger+zinc+finger+peptide,+bound+to+DNA.">Structure of Aart, a designed six-finger zinc finger peptide, bound to DNA.</a> J. Mol. Biol. 363, 405-421 (2006).</li></ol>

作者什么都没有透露。

这里，一步一步协议用于使用细胞渗透性锌指（ZIF）结构域蛋白输送呈现。该ZIF域不降低融合酶的货物34活动;允许生产和收益率几乎相同的那些与未修饰蛋白观察到的蛋白质的纯化;和可运输的蛋白质和酶，用于广泛范围的细胞类型，效率超过传统的细胞穿透肽或蛋白质转导结构域的系统。总之，这些发现表明ZIF域的广泛的潜在介导的直接蛋白输送到细胞内的广泛的应用范围。 <p class="j...

高效，多功能的蛋白质交付策略是许多基础研究和治疗应用的关键。直接输送纯化的蛋白质导入细胞代表一个的最安全和最容易的方法来实现这一点。1,2-不同于依靠从核酸的基因表达的策略，3-5蛋白输送带来插入突变的危险，是独立的细胞转录/翻译机制，并允许立竿见影的效果。然而，由于缺乏简单，可推广的方法赋予细胞渗透活性的蛋白质上经常混淆其直接进入细胞。目前的方法用于促进细胞内蛋白质递送的基于使用的天然存在的6-8或设计的细胞穿透肽，9-12增压转导结构域，13,14纳米颗粒15和脂质体，16病毒样颗粒17,18 </SUP&gt;和聚合物微球材料。19不幸的是，许多这些方法是由低细胞摄取速率，20,21稳定性差，22无意细胞类型特异性，低23内体逃逸属性24和 ​​毒性。25除了受阻，许多蛋白质转导技术减少递送蛋白质的生物活性。14 我们实验室以前表明，锌指核酸酶（ZFN）的蛋白质-嵌合限制性内切酶选自由可编程的Cys 2 -His 2锌指DNA结合蛋白和的FokⅠ限制性内切酶26-28的切割结构域的-是固有小区可渗透的。29这种令人惊奇的细胞穿透活性被证明是定制设计的锌指结构域，即已经成为一个强大的工具用于靶向基因组中的连接DNA结合平台的固有性质工程设计，30-32，被认为是六对蛋白质表面带正电荷的残基的构象的结果。实际上，一些DNA结合蛋白，包括c-Jun的和N-DEK已显示具有穿过细胞膜的固有能力。33最近，我们的实验室扩展了这些结果，并表明，锌的细胞穿透活性手指（ZIF）域，可以利用细胞内蛋白质传递。遗传融合任一个或两个手指ZIF域特异性蛋白货物导致摄取效率超出许多常规细胞穿透肽递送系统34最值得注意的是，ZIF介导的递送没有妥协的稠酶促货物的活性和促进高水平的胞浆交付。总的来说，这些研究结果表明该ZIF域的电位用于促进高效和轻便递送的蛋白质，和宏观的潜在更加多样类型分子进入细胞。 在这里，一个详细的一步一步的协议，就如何落实ZIF技术蛋白交付在哺乳动物细胞中被提出。我们以前建造一套一，二，三，四，五，六指缺乏结合DNA，由于每个α螺旋DNA结合残基的替代能力ZIF域，但能够提供蛋白质进入细胞34（ 图1）。翡翠绿色荧光蛋白（EmGFP）的制备和转导入用两个手指ZIF域HeLa细胞进行说明。这个协议是可扩展的，以几乎能够在大肠杆菌和可溶性表达几乎任何哺乳动物细胞类型的任何蛋白质。提供预期结果，并且还讨论了用于最大化该系统的性能的策略。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>XmaI</td>
			<td>New England Biolabs</td>
			<td>R0180L</td>
			<td></td>
		</tr>
		<tr>
			<td>SacI</td>
			<td>New England Biolabs</td>
			<td>R0156L</td>
			<td></td>
		</tr>
		<tr>
			<td>Expand High Fidelity PCR system</td>
			<td>Roche</td>
			<td>11759078001</td>
			<td></td>
		</tr>
		<tr>
			<td>dNTPs</td>
			<td>New England Biolabs</td>
			<td>N0446S</td>
			<td></td>
		</tr>
		<tr>
			<td>4-20% Tris-Glycine Mini protein gels, 1.5 mm, 10 wells</td>
			<td>Life Technologies</td>
			<td>EC6028BOX</td>
			<td></td>
		</tr>
		<tr>
			<td>2x Laemmli Sample Buffer</td>
			<td>BioRad</td>
			<td>161-0737</td>
			<td></td>
		</tr>
		<tr>
			<td>T4 DNA Ligase</td>
			<td>Life Technologies</td>
			<td>15224-017</td>
			<td></td>
		</tr>
		<tr>
			<td>BL21 (DE3) Competent E. coli</td>
			<td>New England Biolabs</td>
			<td>C2527I</td>
			<td></td>
		</tr>
		<tr>
			<td>IPTG</td>
			<td>Thermo Scientific</td>
			<td>R0391</td>
			<td></td>
		</tr>
		<tr>
			<td>Zinc Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>208086-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>Kanamycin Sulfate</td>
			<td>Fisher Scientific</td>
			<td>BP906-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G8270-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris Base</td>
			<td>Fisher Scientific</td>
			<td>BP152-25</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>S9888-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>DTT</td>
			<td>Fisher Scientific</td>
			<td>PR-V3151&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>PMSF</td>
			<td>Thermo Scientific</td>
			<td>36978</td>
			<td></td>
		</tr>
		<tr>
			<td>Ni-NTA Agarose Resin</td>
			<td>QIAGEN</td>
			<td>30210</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sigma-Aldrich</td>
			<td>G5516-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Imidazole</td>
			<td>Sigma-Aldrich</td>
			<td>I5513-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Amicon Ultra-15 Centrifugal Filter Units</td>
			<td>EMO Millipore</td>
			<td>UFC900324</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Life Technologies</td>
			<td>11966-025</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Life Technologies</td>
			<td>10437-028</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic-Antimycotic&#160;</td>
			<td>Life Technologies</td>
			<td>15240-062</td>
			<td></td>
		</tr>
		<tr>
			<td>24-Well Flat Bottom Plate</td>
			<td>Sigma-Aldrich</td>
			<td>CLS3527-100EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-Lysine</td>
			<td>Sigma-Aldrich</td>
			<td>P7280</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS, No Calcium, No Magnesium</td>
			<td>Life Technologies</td>
			<td>21600010</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparan Sulfate</td>
			<td>Sigma-Aldrich</td>
			<td>H4777</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Life Technologies</td>
			<td>25300054</td>
			<td></td>
		</tr>
		<tr>
			<td>Hela cells</td>
			<td>ATCC</td>
			<td>CCL-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Nano Drop ND-1000 spectrophotometer&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAquick PCR Purification Kit</td>
			<td>QIAGEN</td>
			<td>28104</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAquick Gel Extraction Kit</td>
			<td>QIAGEN</td>
			<td>28704</td>
			<td></td>
		</tr>
	</tbody>
</table>

direct protein delivery to mammalian cells using cell-permeable cys2-his2 zinc-finger domains

Due to their modularity and ability to be reprogrammed to recognize a wide range of DNA sequences, Cys2-His2 zinc-finger DNA-binding domains have emerged as useful tools for targeted genome engineering. Like many other DNA-binding proteins, zinc-fingers also possess the innate ability to cross cell membranes. We recently demonstrated that this intrinsic cell-permeability could be leveraged for intracellular protein delivery. Genetic fusion of zinc-finger motifs leads to efficient transport of protein and enzyme cargo into a broad range of mammalian cell types. Unlike other protein transduction technologies, delivery via zinc-finger domains does not inhibit enzyme activity and leads to high levels of cytosolic delivery. Here a detailed step-by-step protocol is presented for the implementation of zinc-finger technology for protein delivery into mammalian cells. Key steps for achieving high levels of intracellular zinc-finger-mediated delivery are highlighted and strategies for maximizing the performance of this system are discussed.

二指ZIF-EmGFP融合蛋白可以在大肠杆菌中表达大肠杆菌具有&gt; 95％均一性和高的产率（&gt; 25毫克/毫升）（ 图2）。在一般情况下，一个和两个手指ZIF融合蛋白可在数量几乎相同的那些野生型未修饰蛋白的制备。然而，在某些情况下，五元和六手指ZIF融合蛋白不能在产量为下游应用高至足以进行制造。 在37℃下直接应用二指ZIF-EmGFP蛋白到HeLa细胞90分钟?...

Watch this Scientific Journal Video about Direct Protein Delivery to Mammalian Cells Using Cell-permeable Cys2-His2 Zinc-finger Domains at JoVE.com

Direct Protein Delivery to Mammalian Cells Using Cell-permeable Cys2-His2 Zinc-finger Domains

锌指结构域是本质细胞渗透性和能够介导蛋白输送到广泛的哺乳动物细胞类型的。这里，一个详细的一步一步的协议用于实现锌指技术为细胞内蛋白输送呈现。

蛋白质直接送货到哺乳动物细胞利用细胞渗透半胱氨酸<sub&gt; 2</sub&gt; -His<sub&gt; 2</sub&gt;锌指域

Due to their modularity and ability to be reprogrammed to recognize a wide range of DNA sequences, Cys2-His2 zinc-finger DNA-binding domains have emerged ...

direct-protein-delivery-to-mammalian-cells-using-cell-permeable-cys2

Research

JoVE Journal

Biology

Direct Protein Delivery to Mammalian Cells Using Cell-permeable Cys2-His2 Zinc-finger Domains

9.8K 次观看.  The Scripps Research Institute. 锌指结构域是本质细胞渗透性和能够介导蛋白输送到广泛的哺乳动物细胞类型的。这里，一个详细的一步一步的协议用于实现锌指技术为细胞内蛋白输送呈现。 

视频: Direct Protein Delivery to Mammalian Cells Using Cell-permeable Cys2-His2 Zinc-finger Domains

Long-term Imaging Mammalian Cells using Wide-Field Microscopy

长期使用宽视场显微镜的成像哺乳动物细胞

科研

生物学

Trypsinizing and Subculturing Mammalian Cells

As cells reach confluency, they must be subcultured or passaged. Failure to subculture confluent cells results in reduced mitotic index and eventually in cell death. The first step in subculturing is to detach cells from the surface of the primary culture vessel by trypsinization or mechanical means. The resultant cell suspension is then subdivided, or reseeded, into fresh cultures. Secondary cultures are checked for growth and fed periodically, and may be subsequently subcultured to produce tertiary cultures. The time between passaging of cells varies with the cell line and depends on the growth rate.

As cells reach confluency, they must be subcultured or passaged. This video will demonstrate a procedure for subculturing both adherent and suspension cells.

Trypsinizing和传代的哺乳动物细胞

As cells reach confluency, they must be subcultured or passaged. Failure to subculture confluent cells results in reduced mitotic index and eventually in ...

Engineering Cell-permeable Protein

The protein transduction technique enables the direct delivery of biologically active material into mammalian cells [for review see 1,2]. For this one can make use of the translocating ability of so-called cell penetrating peptides (CPPs), also designated as protein transduction domains (PTDs). The TAT-CPP derived from the human immunodeficiency virus type 1 (HIV-1) Tat (trans-activator of transcription) protein has been widely used. The positively charged TAT promotes cell permeability thereby overcoming the barriers of the cellular membrane by endocytosis or/and direct membrane penetration2. In combination with a nuclear localization signal (NLS) fusion proteins are able to enter the nucleus exhibiting functionality. Our video presentation demonstrates, as an exemplification for the engineering of cell-permeable proteins, the construction, production and application of a cell-permeable version of the DNA-modifying enzyme Cre.
Cre is a site-specific recombinase that is able to recognize and recombine 34 base pair loxP sites in mammalian cells in vitro and in vivo. Therefore the Cre/loxP system is widely used to conditionally induce mutations in the genome of living cells3,4. The delivery of active Cre recombinase to cells, however, represents a limitation.
We describe the pSESAME vector system, which allows a direct insertion of the gene-of-interest and provides a platform to rapidly clone different domains and tags used within the vector in a convenient and standardized manner. Rearranging of the different tags has been shown to modify the biochemical properties of the fusion proteins providing a possibility to achieve higher yield and better solubility. We demonstrate how to express and purify recombinant cell-permeant proteins in and from E. coli. The functionality of the recombinant Cre protein is finally validated in cell culture by assessing its intracellular recombinase activity.

Protein transduction enables the direct delivery of biologically active proteins into cells. In contrast to conventional methods such as DNA transfection or viral transduction this non-invasive paradigm allows highly efficient cellular manipulation in a titratable manner circumventing cellular toxicity and the risk of oncogenic transformation by permanent genetic modification.

工程细胞透水蛋白

The protein transduction technique enables the direct delivery of biologically active material into mammalian cells [for review see 1,2]. For this one can ...

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

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

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

液相恢复GELFREE 8100分馏系统使用基于分子量分馏

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

Genome Editing with CompoZr Custom Zinc Finger Nucleases (ZFNs)

Genome editing is a powerful technique that can be used to elucidate gene function and the genetic basis of disease. Traditional gene editing methods such as chemical-based mutagenesis or random integration of DNA sequences confer indiscriminate genetic changes in an overall inefficient manner and require incorporation of undesirable synthetic sequences or use of aberrant culture conditions, potentially confusing biological study. By contrast, transient ZFN expression in a cell can facilitate precise, heritable gene editing in a highly efficient manner without the need for administration of chemicals or integration of synthetic transgenes.
Zinc finger nucleases (ZFNs) are enzymes which bind and cut distinct sequences of double-stranded DNA (dsDNA). A functional CompoZr ZFN unit consists of two individual monomeric proteins that bind a DNA "half-site" of approximately 15-18 nucleotides (see Figure 1). When two ZFN monomers "home" to their adjacent target sites the DNA-cleavage domains dimerize and create a double-strand break (DSB) in the DNA.1 Introduction of ZFN-mediated DSBs in the genome lays a foundation for highly efficient genome editing. Imperfect repair of DSBs in a cell via the non-homologous end-joining (NHEJ) DNA repair pathway can result in small insertions and deletions (indels). Creation of indels within the gene coding sequence of a cell can result in frameshift and subsequent functional knockout of a gene locus at high efficiency.2 While this protocol describes the use of ZFNs to create a gene knockout, integration of transgenes may also be conducted via homology-directed repair at the ZFN cut site.
The CompoZr Custom ZFN Service represents a systematic, comprehensive, and well-characterized approach to targeted gene editing for the scientific community with ZFN technology. Sigma scientists work closely with investigators to 1) perform due diligence analysis including analysis of relevant gene structure, biology, and model system pursuant to the project goals, 2) apply this knowledge to develop a sound targeting strategy, 3) then design, build, and functionally validate ZFNs for activity in a relevant cell line. The investigator receives positive control genomic DNA and primers, and ready-to-use ZFN reagents supplied in both plasmid DNA and in-vitro transcribed mRNA format. These reagents may then be delivered for transient expression in the investigator&#x2019;s cell line or cell type of choice. Samples are then tested for gene editing at the locus of interest by standard molecular biology techniques including PCR amplification, enzymatic digest, and electrophoresis. After positive signal for gene editing is detected in the initial population, cells are single-cell cloned and genotyped for identification of mutant clones/alleles.

Genome Editing with CompoZr Custom Zinc Finger Nucleases ZFNs

The CompoZr Custom Zinc-Finger Nuclease (ZFN) Service enables precise genome editing in any organism or cell line at any locus defined by the user. This article describes the process for the design, manufacture, validation and implementation of the CompoZr Custom ZFN Service.

基因组编辑与的CompoZr自锌指核酸酶（ZFNs）

Genome editing is a powerful technique that can be used to elucidate gene function and the genetic basis of disease. Traditional gene editing methods such ...

siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that regulates diverse biological pathways including the hypoxia response, proteostasis, the DNA damage response and transcription.&#160; To better understand how UBLs regulate pathways relevant to human disease, we have compiled a human siRNA &#8220;ubiquitome&#8221; library consisting of 1,186 siRNA duplex pools targeting all known and predicted components of UBL system pathways. This library can be screened against a range of cell lines expressing reporters of diverse biological pathways to determine which UBL components act as positive or negative regulators of the pathway in question.&#160; Here, we describe a protocol utilizing this library to identify ubiquitome-regulators of the HIF1A-mediated cellular response to hypoxia using a transcription-based luciferase reporter.&#160; An initial assay development stage is performed to establish suitable screening parameters of the cell line before performing the screen in three stages: primary, secondary and tertiary/deconvolution screening. &#160;The use of targeted over whole genome siRNA libraries is becoming increasingly popular as it offers the advantage of reporting only on members of the pathway with which the investigators are most interested.&#160; Despite inherent limitations of siRNA screening, in particular false-positives caused by siRNA off-target effects, the identification of genuine novel regulators of the pathways in question outweigh these shortcomings, which can be overcome by performing a series of carefully undertaken control experiments.

Here, we describe a methodology to perform a targeted siRNA &#8220;ubiquitome&#8221; screen to identify novel ubiquitin and ubiquitin-like regulators of the HIF1A-mediated cellular response to hypoxia.&#160; This can be adapted to any biological pathway where a robust read out of reporter activity is available.

siRNA的筛选识别泛素和泛素样系统压力表生物学途径在培养的哺乳动物细胞

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that ...

Identification of Protein Interaction Partners in Mammalian Cells Using SILAC-immunoprecipitation Quantitative Proteomics

Quantitative proteomics combined with immuno-affinity purification, SILAC immunoprecipitation, represent a powerful means for the discovery of novel protein:protein interactions. By allowing the accurate relative quantification of protein abundance in both control and test samples, true interactions may be easily distinguished from experimental contaminants. Low affinity interactions can be preserved through the use of less-stringent buffer conditions and remain readily identifiable. This protocol discusses the labeling of tissue culture cells with stable isotope labeled amino acids, transfection and immunoprecipitation of an affinity tagged protein of interest, followed by the preparation for submission to a mass spectrometry facility. This protocol then discusses how to analyze and interpret the data returned from the mass spectrometer in order to identify cellular partners interacting with a protein of interest. As an example this technique is applied to identify proteins binding to the eukaryotic translation initiation factors: eIF4AI and eIF4AII.

SILAC immunoprecipitation experiments represent a powerful means for discovering novel protein:protein interactions. By allowing the accurate relative quantification of protein abundance in both control and test samples, true interactions may be easily distinguished from experimental contaminants, and low affinity interactions preserved through use of less-stringent buffer conditions.

的蛋白质相互作用的合作伙伴在哺乳动物细胞中使用SILAC-免疫定量蛋白质组学鉴定

Quantitative proteomics combined with immuno-affinity purification, SILAC immunoprecipitation, represent a powerful means for the discovery of novel ...

Zinc-finger Nuclease Enhanced Gene Targeting in Human Embryonic Stem Cells

One major limitation with current human embryonic stem cell (ESC) differentiation protocols is the generation of heterogeneous cell populations.&#160;These cultures contain the cells of interest, but are also contaminated with undifferentiated ESCs, non-neural derivatives and other neuronal subtypes.&#160; This limits their use in in vitro and in vivo applications, such as in vitro modeling for drug discovery or cell replacement therapy. To help overcome this, reporter cell lines, which offer a means to visualize, track and isolate cells of interest, can be engineered.&#160;However, to achieve this in human embryonic stem cells via conventional homologous recombination is extremely inefficient.&#160;This protocol describes targeting of the Pituitary homeobox 3 (PITX3) locus in human embryonic stem cells using custom designed zinc-finger nucleases, which introduce site-specific double-strand DNA breaks, together with a PITX3-EGFP-specific DNA donor vector. Following the generation of the PITX3 reporter cell line, it can then be differentiated using published protocols for use in studies such as in vitro Parkinson&#8217;s disease modeling or cell replacement therapy.

Reporter cell lines offer a means to visualize, track and isolate cells of interest from heterogeneous populations.&#160;However, gene-targeting using conventional homologous recombination in human embryonic stem cells is extremely inefficient. Herein, we describe targeting CNS midbrain specific transcription factor PITX3 locus with EGFP using zinc-finger nuclease enhanced homologous recombination.

锌指核酸酶增强基因打靶的人胚胎干细胞

One major limitation with current human embryonic stem cell (ESC) differentiation protocols is the generation of heterogeneous cell ...

Examination of Proteins Bound to Nascent DNA in Mammalian Cells Using  BrdU-ChIP-Slot-Western Technique

Histone deacetylases 1 and 2 (HDAC1,2) localize to the sites of DNA replication. In the previous study, using a selective inhibitor and a genetic knockdown system, we showed novel functions for HDAC1,2 in replication fork progression and nascent chromatin maintenance in mammalian cells. Additionally, we used a BrdU-ChIP-Slot-Western technique that combines chromatin immunoprecipitation (ChIP) of bromo-deoxyuridine (BrdU)-labeled DNA with slot blot and Western analyses to quantitatively measure proteins or histone modification associated with nascent DNA.
Actively dividing cells were treated with HDAC1,2 selective inhibitor or transfected with siRNAs against Hdac1 and Hdac2 and then newly synthesized DNA was labeled with the thymidine analog bromodeoxyuridine (BrdU). The BrdU labeling was done at a time point when there was no significant cell cycle arrest or apoptosis due to the loss of HDAC1,2 functions. Following labeling of cells with BrdU, chromatin immunoprecipitation (ChIP) of histone acetylation marks or the chromatin-remodeler was performed with specific antibodies. BrdU-labeled input DNA and the immunoprecipitated (or ChIPed) DNA was then spotted onto a membrane using the slot blot technique and immobilized using UV. The amount of nascent DNA in each slot was then quantitatively assessed using Western analysis with an anti-BrdU antibody. The effect of loss of HDAC1,2 functions on the levels of newly synthesized DNA-associated histone acetylation marks and chromatin remodeler was then determined by normalizing the BrdU-ChIP signal obtained from the treated samples to the control samples.

In this protocol, we describe a novel BrdU-ChIP-Slot-Western technique to examine proteins and histone modifications associated with newly synthesized or nascent DNA.

考试蛋白在哺乳动物细胞中绑定到新生的DNA使用的BrdU芯片插槽西方技术

Histone deacetylases 1 and 2 (HDAC1,2) localize to the sites of DNA replication. In the previous study, using a selective inhibitor and a genetic ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

We present a software solution for semi-automated tracking of relative protein concentration along the length of dynamic cellular protrusions.

软件辅助跟踪动态细胞突变中蛋白质浓度的图形用户界面

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...