Name: cell fractionation of u937 cells by isopycnic density gradient purification
Uploaded: 2021-08-12T13:00:00
Description: Watch this Scientific Journal Video about Cell Fractionation of U937 Cells by Isopycnic Density Gradient Purification at JoVE.com

这项工作得到了NIH R15-HL135675-01和NIH 2 R15-HL135675-02对T.J.L的支持。

1. 制备缓冲液和试剂
<ol><li>准备磷酸酶和蛋白酶抑制剂的新鲜溶液。<ol><li>将 17.4 mg 苯甲磺酰氟 （PMSF） 加入 1 mL 100% 乙醇中以制备 100 mM 储备液。 注意：处理PMSF时应穿戴防护设备，因为摄入或吸入以及接触皮肤或眼睛时是危险的。对眼睛和皮肤有腐蚀性。</li>
		<li>根据制造商的说明，准备市售蛋白酶抑制剂混合物（100x）。</li>
		<li>将 91.9 mg 原钒酸钠 （SOV） 加入 1 mL 去离子水中以制备 500 mM ?...

<ol>
	<li>Baghirova, S., Hughes, B. G., Hendzel, M. J., Schulz, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sequential+fractionation+and+isolation+of+subcellular+proteins+from+tissue+or+cultured+cells.">Sequential fractionation and isolation of subcellular proteins from tissue or cultured cells.</a> MethodsX. 2, 440-445 (2015).</li><li>Il Hwang, S., Han, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subcellular+fractionation+for+identification+of+biomarkers:+Serial+detergent+extraction+by+subcellular+accessibility+and+solubility.">Subcellular fractionation for identification of biomarkers: Serial detergent extraction by subcellular accessibility and solubility.</a> Methods in Molecular Biology. 1002, 25-35 (2013).</li><li>Clayton, D. A., Shadel, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+mitochondria+from+cells+and+tissues.">Isolation of mitochondria from cells and tissues.</a> Cold Spring Harbor Protocols. 2014 (10), 1040-1041 (2014).</li><li>Stimpson, S. E., Coorssen, J. R., Myers, 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=Optimal+isolation+of+mitochondria+for+proteomic+analyses.">Optimal isolation of mitochondria for proteomic analyses.</a> Analytical Biochemistry. 475, 1-3 (2015).</li><li>Sundstr&#246;m, C., Nilsson, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+and+characterization+of+a+human+histiocytic+lymphoma+cell+line+(U-937).">Establishment and characterization of a human histiocytic lymphoma cell line (U-937).</a> International Journal of Cancer. 17 (5), 565-577 (1976).</li><li>Hodge, T., Colombini, M. <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+metabolite+flux+through+voltage-gating+of+VDAC+channels.">Regulation of metabolite flux through voltage-gating of VDAC channels.</a> Journal of Membrane Biology. 157 (3), 271-279 (1997).</li><li>Therien, A. G., Blostein, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+sodium+pump+regulation.+American+journal+of+physiology.">Mechanisms of sodium pump regulation. American journal of physiology.</a> Cell physiology. 279 (3), 541-566 (2000).</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> Proceedings of the National Academy of Sciences of the United States of the America. 76 (9), 4350-4354 (1979).</li><li>Barber, R. D., Harmer, D. W., Coleman, R. A., Clark, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GAPDH+as+a+housekeeping+gene:+analysis+of+GAPDH+mRNA+expression+in+a+panel+of+72+human+tissues.">GAPDH as a housekeeping gene: analysis of GAPDH mRNA expression in a panel of 72 human tissues.</a> Physiological Genomics. 21 (3), 389-395 (2005).</li><li>Bradbury, E. M., Cary, P. D., Crane-Robinson, C., Rattle, H. W. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conformations+and+interactions+of+histones+and+their+role+in+chromosome+structure.">Conformations and interactions of histones and their role in chromosome structure.</a> Annals of the New York Academy of Sciences. 222, 266-289 (1973).</li><li>McCaig, W. D., Deragon, M. A., Haluska, R. J., Hodges, A. L., Patel, P. S., LaRocca, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+fractionation+of+U937+cells+in+the+absence+of+high-speed+centrifugation.">Cell fractionation of U937 cells in the absence of high-speed centrifugation.</a> Journal of Visualized Experiments. (143), e59022 (2019).</li><li>McCaig, W. D., 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=Hyperglycemia+potentiates+a+shift+from+apoptosis+to+RIP1-dependent+necroptosis.">Hyperglycemia potentiates a shift from apoptosis to RIP1-dependent necroptosis.</a> Cell Death Discovery. 4, 55 (2018).</li></ol>

该方法是先前发表的亚细胞分级分离方法的修改版本，无需使用高速离心11。这种改进的方法需要更专业的设备才能获得最佳结果，但更全面且可重复性一致。
由于无法分离线粒体和膜样品以分析坏死性凋亡期间的蛋白质定位，因此有必要开发初始方案12。尝试使用大多数市售试剂盒中发现的完全基于洗涤剂的方法，导致含有质膜和细胞中?...

An erratum was issued for:&#160;<a href="https://www.jove.com/t/62119">Cell Fractionation of U937 Cells by Isopycnic Density Gradient Purification</a>. The Authors section was&#160;updated.
The Authors section was updated from:
William McCaig1 
Timothy LaRocca1 
1Department of Basic and Clinical Sciences, Albany College of Pharmacy and Health Sciences
to:
William D. McCaig1 
Matthew A. Deragon1 
Phillip V. Truong1 
Angeleigh R. Knapp1 
Keven J. Hughes1 
Timothy J. LaRocca1 
1Department of Basic and Clinical Sciences, Albany College of Pharmacy and Health Sciences

An erratum was issued for:&#160;<a href="https://www.jove.com/t/62119">Cell Fractionation of U937 Cells by Isopycnic Density Gradient Purification</a>. The Authors section was&#160;updated.

Erratum: Cell Fractionation of U937 Cells by Isopycnic Density Gradient Purification

An erratum was issued for:&#160;<a href="https://www.jove.com/t/62119">Cell Fractionation of U937 Cells by Isopycnic Density Gradient Purification</a>. The Authors section was&#160;updated.
The Authors section was updated from:
William D. McCaig1 
Matthew A. Deragon1 
Phillip V. Truong1 
Angeleigh R. Knapp1 
Keven J. Hughes1 
Timothy J. LaRocca1 
1Department of Basic and Clinical Sciences, Albany College of Pharmacy and Health Sciences
to:
William D. McCaig1 
Matthew A. Deragon1 
Phillip V. Truong1 
Angeleigh R. Knapp1 
Timothy J. LaRocca1 
1Department of Basic and Clinical Sciences, Albany College of Pharmacy and Health Sciences

亚细胞分级分离是通过多种方法裂解细胞并将其分离成其组成成分的过程。研究人员可以使用这种技术来确定哺乳动物细胞中的蛋白质定位或富集否则无法检测到的低丰度蛋白质。虽然目前存在亚细胞分离的方法，也可以购买商业试剂盒，但它们受到该程序试图克服的几个限制。大多数细胞分级分离方法完全基于洗涤剂1，2，依赖于使用含有越来越多的去垢剂的缓冲液来溶解不同的细胞成分。虽然这种方法快速方便，但它会导致不纯的馏分。这些旨在使研究人员能够轻松分离细胞的一种或两种成分，但还不够复杂，无法同时从样品中分离出多个亚细胞组分。仅依靠洗涤剂通常会导致膜封闭的细胞器和质膜被不加选择地溶解，从而使这些成分的分离变得困难。使用这些试剂盒的另一个复杂因素是研究人员无法针对特定应用对其进行更改/优化，因为大多数组件都是专有配方。最后，这些试剂盒可能非常昂贵，使用次数有限，因此对于较大样品来说不太理想。
尽管有不依赖去垢剂的线粒体分离试剂盒，但它们并非设计用于分离质膜，并且产生的样品量明显低于标准分离方案3，4。虽然差速离心方法更耗时，但它们通常会产生不同的馏分，而仅使用基于洗涤剂的试剂盒无法获得1。无需单独使用增溶去垢剂的分离还允许使用超速离心和等密度梯度进一步纯化，从而减少交叉污染。该分级分离方案展示了使用基于去垢剂和高速离心的方法的组合从U937单核细胞中分离亚细胞级分。该方法将有助于分离哺乳动物细胞的核、细胞质、线粒体和质膜成分，而馏分之间的污染最小。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Benzonase Nuclease</td>
			<td>Sigma-Aldrich</td>
			<td>E1014</td>
			<td></td>
		</tr>
		<tr>
			<td>Bullet Blender Tissue Homogenizer</td>
			<td>Next Advance</td>
			<td>61-BB50-DX</td>
			<td></td>
		</tr>
		<tr>
			<td>digitonin</td>
			<td>Sigma</td>
			<td>D141</td>
			<td></td>
		</tr>
		<tr>
			<td>end-over-end rotator</td>
			<td>ThermoFisher</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid (EDTA)</td>
			<td>Sigma</td>
			<td>E9884</td>
			<td></td>
		</tr>
		<tr>
			<td>ethylene glycol-bis(&#946;-aminoethyl ether)-N,N,N&#39;,N&#39;-tetraacetic acid (EGTA)</td>
			<td>Sigma</td>
			<td>E3889</td>
			<td></td>
		</tr>
		<tr>
			<td>GAPDH (14C10)&#160;</td>
			<td>Cell Signalling Technologies</td>
			<td>2118</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>VWR</td>
			<td>97064-360</td>
			<td></td>
		</tr>
		<tr>
			<td>Hexylene glycol</td>
			<td>Sigma</td>
			<td>68340</td>
			<td></td>
		</tr>
		<tr>
			<td>Igepal</td>
			<td>Sigma</td>
			<td>I7771</td>
			<td>Non-ionic, non-denaturing detergent</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma</td>
			<td>P9333</td>
			<td></td>
		</tr>
		<tr>
			<td>Mannitol</td>
			<td>Sigma</td>
			<td>M9647</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma</td>
			<td>M8266</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma</td>
			<td>S9888</td>
			<td></td>
		</tr>
		<tr>
			<td>Na, K-ATPase a1 (D4Y7E)</td>
			<td>Cell Signalling Technologies</td>
			<td>23565</td>
			<td></td>
		</tr>
		<tr>
			<td>Open-Top Polyclear Tubes, 16 x 52 mm</td>
			<td>Seton Scientific</td>
			<td>7048</td>
			<td></td>
		</tr>
		<tr>
			<td>OptiPrep (Iodixanol)&#160;Density Gradient Medium</td>
			<td>Sigma</td>
			<td>D1556-250ML</td>
			<td></td>
		</tr>
		<tr>
			<td>phenylmethanesulfonyl fluoride (PMSF)</td>
			<td>Sigma</td>
			<td>P7626</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease Inhibitor Cocktail, General Use</td>
			<td>VWR</td>
			<td>M221-1ML</td>
			<td></td>
		</tr>
		<tr>
			<td>refrigerated centrifuge</td>
			<td>ThermoFisher</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>S50-ST Swinging Bucket Rotor</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium dodecyl sulfate (SDS)</td>
			<td>Sigma</td>
			<td>436143</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium deoxycholate</td>
			<td>Sigma</td>
			<td>D6750</td>
			<td></td>
		</tr>
		<tr>
			<td>sodium orthovanadate (SOV)</td>
			<td>Sigma</td>
			<td>567540</td>
			<td></td>
		</tr>
		<tr>
			<td>sonicator</td>
			<td>ThermoFisher</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sorvall MX120 Plus Micro-Ultracentrifuge</td>
			<td>ThermoFisher</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Stainless Steel Beads 3.2 mm</td>
			<td>Next Advance</td>
			<td>SSB32</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma</td>
			<td>S0389</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-buffered Saline (TBS)</td>
			<td>VWR</td>
			<td>97062-370</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td></td>
			<td></td>
			<td>non-ionic detergent in western blotting buffers</td>
		</tr>
		<tr>
			<td>VDAC (D73D12)</td>
			<td>Cell Signalling Technologies</td>
			<td>4661</td>
			<td></td>
		</tr>
	</tbody>
</table>

cell fractionation of u937 cells by isopycnic density gradient purification

该协议描述了使用洗涤剂，机械裂解和等密度梯度离心的组合从哺乳动物细胞中获得亚细胞蛋白级分的方法。该程序的主要优点是它不依赖于仅使用增溶去垢剂来获得亚细胞级分。这使得可以将质膜与细胞的其他膜结合细胞器分离。该程序将有助于使用可重复，可扩展和选择性的方法确定细胞中的蛋白质定位。该方法已成功用于从人单核细胞系U937中分离胞质蛋白、核蛋白、线粒体蛋白和质膜蛋白。尽管针对该细胞系进行了优化，但该过程可以作为其他细胞系亚细胞分离的合适起点。讨论了该程序的潜在陷阱以及如何避免它们，以及可能需要考虑其他细胞系的改变。

该过程的示意图流程图（图1）直观地总结了成功分级分离悬浮生长的U9375 细胞的步骤。从等密度梯度顶部以等体积（1 mL）收集的级分显示了线粒体和膜组分的纯化（图2）。利用针对VDAC（一种定位于线粒体外膜6的蛋白质）的抗体，表明线粒体部分迁移到25%和30%碘沙醇（v / v）部分（图2A）。使?...

Watch this Scientific Journal Video about Cell Fractionation of U937 Cells by Isopycnic Density Gradient Purification at JoVE.com

Cell Fractionation of U937 Cells by Isopycnic Density Gradient Purification

该分级分离方案将使研究人员能够从哺乳动物细胞中分离细胞质、核、线粒体和膜蛋白。后两个亚细胞级分通过等密度梯度进一步纯化。

通过等密度梯度纯化对U937细胞进行细胞分级分离

This protocol describes a method to obtain subcellular protein fractions from mammalian cells using a combination of detergents, mechanical lysis, and ...

该协议很重要，因为它提供了一种将四个细胞区室彼此分离的方法，即细胞质，细胞核，线粒体和膜。不仅如此，它还是一种可扩展、可重复的程序，具有可靠的结果。该技术的主要优点是分离四个细胞区室，洗涤剂的使用最少，从而实现更可重复、更可靠的分馏过程。对于胞质蛋白分离，在RPMI 1640中用10%胎牛血清在37摄氏度和5%二氧化碳下培养U937细胞，最终总共6个乘10至第八个细胞。离心培养物，并将细胞沉淀重悬于室温PBS中至终浓度为每毫升4×10至第六个细胞，轻轻移液以分解团块。第二次离心后，将沉淀重悬于冰冷的裂解缓冲液A中，终浓度为每毫升2×10至第七个细胞。将7.5毫升细胞加入冰上的锥形管中用于下游核蛋白提取，并通过离心沉淀剩余的细胞。将沉淀重悬于细胞质分离缓冲液中，终浓度为每毫升2×10至第七个细胞，轻轻移液以分解团块，然后将细胞在4摄氏度下孵育20分钟，并结束旋转。在孵育结束时，离心细胞悬液并将上清液转移到干净的离心管中。离心上清液以沉淀细胞碎片。离心后，将上清液转移到新的离心管中，并重复离心序列，直到观察到无沉淀。当样品清除碎片时，将含有胞质级分的上清液在 4 摄氏度下储存长达一个月。然后将储存的细胞沉淀重悬于裂解缓冲液A中，至终浓度为每毫升4×10至第六个细胞。对于细胞匀浆，离心细胞悬液并丢弃上清液以去除多余的洋地黄皂苷和收缩期污染物。将沉淀重悬于冰冷细胞匀浆缓冲液中，终浓度为每毫升4×10至第六个细胞，在冰上孵育30分钟。同时，对于基于珠子的机械裂解，将30克预洗的不锈钢3.2毫米珠子加入15毫升裂解缓冲液B中，放入50毫升裙边管中的冰上冷却。在孵育结束时，用细胞悬液替换裙边管中的缓冲液，并以第八速度混合细胞五分钟。裂解后，将匀浆转移到干净的管中。离心匀浆，然后将上清液转移到新管中。要从样品中去除任何残留的碎屑，请按照指示将匀浆离心三次，每次离心后将上清液转移到新管中。最后一次离心后，为了分离粗线粒体级分，将上清液转移到新管中进行离心。离心后，将上清液转移到新管中，并将含线粒体级分的粗沉淀放在冰上。为了分离膜级分，离心收集的上清液，丢弃上清液后将含有粗膜级分的沉淀放在冰上。对于等密度梯度纯化，将粗线粒体和膜级分沉淀重悬于200微升裂解缓冲液B和1.8毫升碘沙醇中。要为每个样品创建不连续的碘沙醇梯度，首先，将一毫升 15% 碘沙醇添加到每个样品一个 8 毫升开顶薄壁超速离心管的底部。接下来，在每个管中依次在15%碘沙醇层下方覆盖一毫升20%碘沙醇，一毫升25%碘沙醇，一毫升30%碘沙醇和一毫升35%碘沙醇。在一个梯度的底层下加入两毫升粗线粒体颗粒悬浮液，在第二梯度的底层下加入两毫升粗膜颗粒悬浮液。小心地将一毫升10%碘沙醇层在每个梯度的顶部，并在通过密度梯度离心纯化线粒体和膜组分之前，根据需要添加10%碘沙醇来平衡10微克内的试管。在分离结束时，使用针刺穿薄壁管的侧面，以收集每个样品的可见条带。对于核蛋白分离，离心悬浮在裂解缓冲液A中的7.5毫升等分试样细胞，并通过移液和涡旋将沉淀重悬于800微升冰冷细胞增溶缓冲液中。将悬浮液在冰上孵育30分钟以破坏血浆和细胞器膜，同时保护核蛋白。在孵育结束时，离心细胞悬液并通过轻轻移液到800微升冰冷的核裂解缓冲液中重新悬浮沉淀，每微升Benzonase补充一个单位。将混合物在四摄氏度的末端旋转器上孵育以破坏核膜。30 分钟后，以 20% 的功率超声处理混合物三次，每次 5 秒，脉冲之间暂停 5 秒以剪切核酸。最后一次超声处理后，离心匀浆并收集上清液作为核级分，而不会干扰沉淀。密度梯度纯化后每个级分的蛋白质印迹显示线粒体级分定位到25%和30%碘沙醇层，膜级分定位到10%和15%碘沙醇层。蛋白质血液分析还证实，每个分离的样品都是纯净的，不受细胞其他部分蛋白质的污染。此外，对每个样品的条带强度进行密度分析证实了数据的重现性和统计意义。分级分离执行不当会导致细胞成分交叉污染。例如，胞质级分中高浓度的组蛋白H3可能是由于胞质级分的澄清不当造成的。等密度纯化过程中的失败会导致膜组分污染。在蛋白质免疫印迹分析之前进行蛋白质定量测定以确认馏分确实含有蛋白质以允许基于蛋白质量的凝胶上样，并确认该程序已正确执行，这可能是有用的。至关重要的是，细胞质部分不含沉淀。可以按照此过程进行蛋白质印迹或ELISA，以允许您在特定条件下分析不同蛋白质的亚细胞位置。这项技术使我们能够分析高血糖条件下不同细胞死亡因子的运输。因此，对我们来说，这有助于阐明从细胞凋亡到坏死的高血糖转变的机制。

Research

JoVE Journal

Biochemistry

Tandem Affinity Purification of Protein Complexes from Eukaryotic Cells

从真核细胞蛋白质复合体串联亲和纯化

The purification of active protein-protein and protein-nucleic acid complexes is crucial for the characterization of enzymatic activities and de novo ...

科研

生物化学

Purification of Biotinylated Cell Surface Proteins from Rhipicephalus microplus Epithelial Gut Cells

Purification of Biotinylated Cell Surface Proteins from Rhipicephalus microplus Epithelial Gut Cells

生物素化细胞表面蛋白的纯化<emRigicephalus microplus</em&gt;上皮细胞

Rhipicephalus microplus &#8211; the cattle tick &#8211; is the most significant ectoparasite in terms of economic impact on livestock as a vector of ...

Purification of Hepatocytes and Sinusoidal Endothelial Cells from Mouse Liver Perfusion

小鼠肝灌注肝细胞和正弦内皮细胞的纯化

This protocol demonstrates a method for obtaining high yield and viability for mouse hepatocytes and sinusoidal endothelial cells (SECs) suitable for ...

Fractionation for Resolution of Soluble and Insoluble Huntingtin Species

可溶性和不溶性杭丁顿蛋白的分离分解

The accumulation of misfolded proteins is central to pathology in Huntington's disease (HD) and many other neurodegenerative disorders. Specifically, a ...

In Situ Measurement and Correlation of Cell Density and Light Emission of Bioluminescent Bacteria

In Situ Measurement and Correlation of Cell Density and Light Emission of Bioluminescent Bacteria

就地生物发光细菌细胞密度与光发射的测量与相关性研究

There is a considerable number of bacterial species capable of emitting light. All of them share the same gene cluster, namely the lux operon. Despite ...

The Identification of Sea Lamprey Pheromones Using Bioassay-Guided Fractionation

利用生物测定引导分馏法鉴定海洋鳗信息素

Bioassay-guided fractionation is an iterative approach that uses the results of physiological and behavioral bioassays to guide the isolation and ...

Cell Fractionation of U937 Cells in the Absence of High-speed Centrifugation

高速离心条件下 u937 细胞的细胞分裂

In this protocol we detail a method to obtain subcellular fractions of U937 cells without the use of ultracentrifugation or indiscriminate detergents. ...

Measuring Interactions between Fluorescent Probes and Lignin in Plant Sections by sFLIM Based on Native Autofluorescence

基于原生自荧光的sFLIM测量植物部分荧光探针和木质素之间的相互作用

In lignocellulosic biomass (LB), the activity of enzymes is limited by the appearance of non-specific interactions with lignin during the hydrolysis ...

Density Gradient Ultracentrifugation for Investigating Endocytic Recycling in Mammalian Cells

密度梯度超速离心研究哺乳动物细胞内吞循环

Endosomal trafficking is an essential cellular process that regulates a broad range of biological events. Proteins are internalized from the plasma ...

Polysome Profiling without Gradient Makers or Fractionation Systems

无需梯度测定器或分馏系统的多聚体分析

Polysome fractionation by sucrose density gradient centrifugation is a powerful tool that can be used to create ribosome profiles, identify specific mRNAs ...