Name: isolation and analysis of plasma lipoproteins by ultracentrifugation
Uploaded: 2021-01-28T15:00:00
Description: Watch this Scientific Journal Video about Isolation and Analysis of Plasma Lipoproteins by Ultracentrifugation at JoVE.com

这项工作部分得到了JSPS KAKENHI赠款编号JP 20K08858、中国国家自然科学基金（第81941001号和81770457号）的研究资助。

兔子研究的所有程序均经山梨大学机构动物护理和使用委员会批准（批准编号：A28-39）。
1. 血浆与兔血分离
<ol><li>准备 1.5 mL 微管，其中含有 15μL 的 0.5 M EDTA （pH 8.0）， 用于采血。</li>
	<li>将兔子放入约束器中，用22量针刺穿动脉中间动脉，并将血液收集到管子中。轻轻地将血液与EDTA混合，放在冰上。</li>
	<li>在4°C下将血管在1，500 x g 上集中20分钟，并将血浆收集到?...

<ol>
	<li>Gordon, T., Castelli, W. P., Hjortland, M. C., Kannel, W. B., Dawber, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+density+lipoprotein+as+a+protective+factor+against+coronary+heart+disease:+The+Framingham+study.">High density lipoprotein as a protective factor against coronary heart disease: The Framingham study.</a> The American Journal of Medicine. 62 (5), 707-714 (1977).</li><li>Baigent, C., 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=Efficacy+and+safety+of+cholesterol-lowering+treatment:+prospective+meta-analysis+of+data+from+90,056+participants+in+14+randomised+trials+of+statins.">Efficacy and safety of cholesterol-lowering treatment: prospective meta-analysis of data from 90,056 participants in 14 randomised trials of statins.</a> Lancet. 366 (9493), 1267-1278 (2005).</li><li>Nauck, M., Warnick, G. R., Rifai, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+Measurement+of+LDL-Cholesterol:+A+Critical+Assessment+of+Direct+Measurement+by+Homogeneous+Assays+versus+Calculation.">Methods for Measurement of LDL-Cholesterol: A Critical Assessment of Direct Measurement by Homogeneous Assays versus Calculation.</a> Clinical Chemistry. 48 (2), 236-254 (2002).</li><li>Warnick, G. R., Nauck, M., Rifai, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+of+Methods+for+Measurement+of+HDL-Cholesterol:+From+Ultracentrifugation+to+Homogeneous+Assays.">Evolution of Methods for Measurement of HDL-Cholesterol: From Ultracentrifugation to Homogeneous Assays.</a> Clinical Chemistry. 47 (9), 1579-1596 (2001).</li><li>Havel, R. J., Eder, H. A., Bragdon, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+distribution+and+chemical+composition+of+ultracentrifugally+separated+lipoproteins+in+human+serum.">The distribution and chemical composition of ultracentrifugally separated lipoproteins in human serum.</a> The Journal of Clinical Investigation. 34 (9), 1345-1353 (1955).</li><li>Hatch, F. T., Lees, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Practical+Methods+for+Plasma+Lipoprotein+Analysis.">Practical Methods for Plasma Lipoprotein Analysis.</a> Advances in Lipid Research. 6, 1-68 (1968).</li><li>Lindgren, F. T., Adamson, G. L., Jenson, L. C., Wood, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid+and+lipoprotein+measurements+in+a+normal+adult+American+population.">Lipid and lipoprotein measurements in a normal adult American population.</a> Lipids. 10 (12), 750-756 (1975).</li><li>Fan, 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=Overexpression+of+hepatic+lipase+in+transgenic+rabbits+leads+to+a+marked+reduction+of+plasma+high+density+lipoproteins+and+intermediate+density+lipoproteins.">Overexpression of hepatic lipase in transgenic rabbits leads to a marked reduction of plasma high density lipoproteins and intermediate density lipoproteins.</a> Proceedings of the National Academy of Sciences of the United States of America. 91, 8724-8728 (1994).</li><li>Wang, Y., 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=Human+Apolipoprotein+A-II+Protects+Against+Diet-Induced+Atherosclerosis+in+Transgenic+Rabbits.">Human Apolipoprotein A-II Protects Against Diet-Induced Atherosclerosis in Transgenic Rabbits.</a> Arteriosclerosis, Thrombosis, and Vascular Biology. 33 (2), 224-231 (2013).</li><li>Niimi, M., 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=ApoE+knockout+rabbits:+A+novel+model+for+the+study+of+human+hyperlipidemia.">ApoE knockout rabbits: A novel model for the study of human hyperlipidemia.</a> Atherosclerosis. 245, 187-193 (2016).</li><li>Zhang, 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=Deficiency+of+Cholesteryl+Ester+Transfer+Protein+Protects+Against+Atherosclerosis+in+RabbitsHighlights.">Deficiency of Cholesteryl Ester Transfer Protein Protects Against Atherosclerosis in RabbitsHighlights.</a> Arteriosclerosis, Thrombosis, and Vascular Biology. 37 (6), 1068-1075 (2017).</li><li>Yan, H., 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=Endothelial+Lipase+Exerts+its+Anti-Atherogenic+Effect+through+Increased+Catabolism+of+&#946;-VLDLs.">Endothelial Lipase Exerts its Anti-Atherogenic Effect through Increased Catabolism of &#946;-VLDLs.</a> Journal of Atherosclerosis and Thrombosis. 40 (9), 2095-2107 (2020).</li><li>Fan, 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=Overexpression+of+Lipoprotein+Lipase+in+Transgenic+Rabbits+Inhibits+Diet-induced+Hypercholesterolemia+and+Atherosclerosis.">Overexpression of Lipoprotein Lipase in Transgenic Rabbits Inhibits Diet-induced Hypercholesterolemia and Atherosclerosis.</a> Journal of Biological Chemistry. 276 (43), 40071-40079 (2001).</li><li>Koike, 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=Expression+of+Human+ApoAII+in+Transgenic+Rabbits+Leads+to+Dyslipidemia:+A+New+Model+for+Combined+Hyperlipidemia.">Expression of Human ApoAII in Transgenic Rabbits Leads to Dyslipidemia: A New Model for Combined Hyperlipidemia.</a> Arteriosclerosis, Thrombosis, and Vascular Biology. 29 (12), 2047-2053 (2009).</li><li>Ichikawa, 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=Overexpression+of+lipoprotein+lipase+in+transgenic+rabbits+leads+to+increased+small+dense+LDL+in+plasma+and+promotes+atherosclerosis.">Overexpression of lipoprotein lipase in transgenic rabbits leads to increased small dense LDL in plasma and promotes atherosclerosis.</a> Laboratory investigation; a Journal of Technical Methods and Pathology. 84 (6), 715-726 (2004).</li><li>von Zychlinski, A., Kleffmann, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissecting+the+proteome+of+lipoproteins:+New+biomarkers+for+cardiovascular+diseases.">Dissecting the proteome of lipoproteins: New biomarkers for cardiovascular diseases.</a> Translational Proteomics. 7, 30-39 (2015).</li><li>Yan, H., 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=Apolipoprotein+CIII+Deficiency+Protects+Against+Atherosclerosis+in+Knockout+Rabbits.">Apolipoprotein CIII Deficiency Protects Against Atherosclerosis in Knockout Rabbits.</a> Arteriosclerosis, Thrombosis, and Vascular Biology. 28 (2), 157-168 (2021).</li><li>Kang, I., Park, M., Yang, S. J., Lee, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipoprotein+Lipase+Inhibitor,+Nordihydroguaiaretic+Acid,+Aggravates+Metabolic+Phenotypes+and+Alters+HDL+Particle+Size+in+the+Western+Diet-Fed+db/db+Mice.">Lipoprotein Lipase Inhibitor, Nordihydroguaiaretic Acid, Aggravates Metabolic Phenotypes and Alters HDL Particle Size in the Western Diet-Fed db/db Mice.</a> International Journal of Molecular Sciences. 20 (12), 3057 (2019).</li><li>De Silva, H. V., 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=Overexpression+of+human+apolipoprotein+C-III+in+transgenic+mice+results+in+an+accumulation+of+apolipoprotein+B48+remnants+that+is+corrected+by+excess+apolipoprotein+E.">Overexpression of human apolipoprotein C-III in transgenic mice results in an accumulation of apolipoprotein B48 remnants that is corrected by excess apolipoprotein E.</a> Journal of Biological Chemistry. 269 (3), 2324-2335 (1994).</li><li>Usui, S., Hara, Y., Hosaki, S., Okazaki, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+on-line+dual+enzymatic+method+for+simultaneous+quantification+of+cholesterol+and+triglycerides+in+lipoproteins+by+HPLC.">A new on-line dual enzymatic method for simultaneous quantification of cholesterol and triglycerides in lipoproteins by HPLC.</a> Journal of Lipid Research. 43 (5), 805-814 (2002).</li></ol>

高脂血症是动脉粥样硬化疾病最重要的危险因素之一。因此，对血浆脂蛋白的分析不仅对血脂血症患者的诊断至关重要，而且对研究脂蛋白代谢和动脉粥样硬化的分子机制也具有重要意义。在这项研究中，我们描述了等离子脂蛋白的分离和分析方案，这些协议可以应用于有超中心化的实验室。通过这种方法获得的信息是全面和直接的，因此建议临床和基础研究科学家。分离脂蛋白也可用于研究脂?...

血脂血症是世界上动脉粥样硬化的主要危险因素。高浓度的低密度脂蛋白（LDLs）和低水平的高密度脂蛋白（HDL）与冠心病的高风险（CHD）1，2密切相关。在临床环境中，低密度脂蛋白胆固醇（LDL-C）和高密度脂蛋白胆固醇（HDL-C）都是在临床实验室3、4中使用自动分析仪进行常规测量的。尽管如此，还是有必要详细分析脂蛋白特征，以诊断脂质血症，研究人类和实验动物的脂质代谢和动脉粥样硬化。据报道，分析血浆脂蛋白的几种方法，如超中心化、大小排除色谱[快速蛋白质液体色谱（FPLC）和高性能液体色谱（HPLC）]、阿加罗斯和多丙烯酰胺凝胶的电磷化、核磁共振以及使用多面体和二价沉淀或其他化学品的选择性化学沉淀。20世纪50年代，Havel的研究小组首次提出了由密度定义的利波蛋白的概念，并将其分为低密度脂蛋白（CM）、低密度脂蛋白（VLDL）、中密度脂蛋白（IDL）、LDL和HDL5等，后来，其他组6、7进一步修改了该方法。到目前为止，超中心化是最流行和最可靠的方法，而实用的协议仍然不可用。在本文中，我们试图描述一个易于使用的方案，用于隔离小比例的等离子体使用顺序密度浮动超中心最初描述以前8。隔离七个等离子脂蛋白分数 [VLDL（d=lt;1.006 g/mL）、IDL（d=1.02 g/mL）、LDL（d=1.04 和 1.06 g/mL）、高密度脂蛋白（d=1.08， 1.10 和 1.21 克/mL）]使研究人员能够对脂蛋白及其组成非脊蛋白9、10、11进行广泛分析。完整的连续七密度脂蛋白可用于分析脂蛋白功能，也可用于基于细胞的体外策略。此协议应可用于临床诊断和基础研究。在这里，我们以兔子等离子体为例来证明这项技术，而来自其他物种的等离子体也可以以同样的方式应用。

<table>
	<tbody>
		<tr>
			<td>22-gauge needle</td>
			<td>Terumo</td>
			<td>NN-2232S</td>
			<td>For blood collection</td>
		</tr>
		<tr>
			<td>96-well microplate</td>
			<td>greiner bio-one</td>
			<td>655101</td>
			<td>For lipids measurment</td>
		</tr>
		<tr>
			<td>Anti-apolipoprotein A-I antibody</td>
			<td>LifeSpan BioSciences</td>
			<td>LS-C314186</td>
			<td>For Western blottng, use 1:1,000</td>
		</tr>
		<tr>
			<td>Anti-apolipoprotein B antibody</td>
			<td>ROCKLAND</td>
			<td>600-101-111</td>
			<td>For Western blottng, use 1:1,000</td>
		</tr>
		<tr>
			<td>Anti-apolipoprotein E antibody</td>
			<td>Merck Millipore</td>
			<td>AB947</td>
			<td>For Western blottng, use 1:1,000</td>
		</tr>
		<tr>
			<td>CBB staining kit</td>
			<td>FUJIFILM Wako Pure Chemical</td>
			<td>299-50101</td>
			<td>For apolipoprotein analysis</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>HITACHI</td>
			<td></td>
			<td>himac CF15RN</td>
		</tr>
		<tr>
			<td>Closure</td>
			<td>Spectrum</td>
			<td>132736</td>
			<td>For lipoprotein dialysis</td>
		</tr>
		<tr>
			<td>Dialysis tubing</td>
			<td>FUJIFILM Wako Pure Chemical</td>
			<td>043-30921</td>
			<td>For lipoprotein dialysis, MWCO 14,000</td>
		</tr>
		<tr>
			<td>Dry heat block</td>
			<td>Major Science</td>
			<td>MD-01N</td>
			<td>For SDS-PAGE sample preparation</td>
		</tr>
		<tr>
			<td>ECL Western blotting detection reagents</td>
			<td>GE Healthcare</td>
			<td>RPN2209</td>
			<td>For Western blotting</td>
		</tr>
		<tr>
			<td>Electrophoresis Chamber</td>
			<td>BIO-RAD</td>
			<td></td>
			<td>Mini-PROTEAN Tetra Cell</td>
		</tr>
		<tr>
			<td>Ethylenediamine-N,N,N&#39;,N&#39;-tetraacetic acid (EDTA) disodium salt dihydrate</td>
			<td>FUJIFILM Wako Pure Chemical</td>
			<td>345-01865</td>
			<td>For anticoagulant (0.5 M), for dialysis (1 mM)</td>
		</tr>
		<tr>
			<td>Filter paper</td>
			<td>ADVANTEC</td>
			<td>590</td>
			<td>For Western blotting</td>
		</tr>
		<tr>
			<td>Fixed angle ultracentrifuge rotor</td>
			<td>BECKMAN COULTER</td>
			<td>357656</td>
			<td>TLA-120.2</td>
		</tr>
		<tr>
			<td>Fixing solution</td>
			<td></td>
			<td></td>
			<td>For SDS-PAGE (50% Methanol/ 10% Acetic acid)</td>
		</tr>
		<tr>
			<td>Immun-Blot PVDF menbrane</td>
			<td>BIO-RAD</td>
			<td>1620177</td>
			<td>For Western blotting</td>
		</tr>
		<tr>
			<td>Lumino image analyzer</td>
			<td>GE Healthcare</td>
			<td></td>
			<td>For Western blotting, ImageQuant LAS 4000</td>
		</tr>
		<tr>
			<td>Magnetic stirrer</td>
			<td>ADVANTEC</td>
			<td>SR-304</td>
			<td>For lipoprotein dialysis</td>
		</tr>
		<tr>
			<td>Microplate reader iMARK</td>
			<td>BIO-RAD</td>
			<td></td>
			<td>For lipids measurment</td>
		</tr>
		<tr>
			<td>Microtube</td>
			<td>INA-OPTIKA</td>
			<td>SC-0150</td>
			<td></td>
		</tr>
		<tr>
			<td>Orbital agitator USBDbo</td>
			<td>Stovall Life Science</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Peroxidase congugated anti goat IgG antibody</td>
			<td>Jackson ImmunoResearch</td>
			<td>705-035-003</td>
			<td>For Western blotting, use 1:2,000</td>
		</tr>
		<tr>
			<td>Peroxidase congugated anti mouse IgG antibody</td>
			<td>Jackson ImmunoResearch</td>
			<td>715-035-150</td>
			<td>For Western blotting, use 1:2,000</td>
		</tr>
		<tr>
			<td>Phospholipids assay kit</td>
			<td>FUJIFILM Wako Pure Chemical</td>
			<td>433-36201</td>
			<td>For lipids measurment</td>
		</tr>
		<tr>
			<td>Polycarbonate ultracentrifuge Tubes</td>
			<td>BECKMAN COULTER</td>
			<td>343778</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Bromide</td>
			<td>FUJIFILM Wako Pure Chemical</td>
			<td>168-03475</td>
			<td>For density solution</td>
		</tr>
		<tr>
			<td>Power Supply</td>
			<td>BIO-RAD</td>
			<td></td>
			<td>For SDD-PAGE and Western blotting, PowerPac 300, PowerPac HC</td>
		</tr>
		<tr>
			<td>Protein standards Precidion Plus Protein Dual Xtra</td>
			<td>BIO-RAD</td>
			<td>161-0377</td>
			<td>For SDS-PAGE and Western blotting</td>
		</tr>
		<tr>
			<td>Rabbit restrainer</td>
			<td>Natsume Seisakusho</td>
			<td>KN-318</td>
			<td>For blood collection</td>
		</tr>
		<tr>
			<td>Rotor</td>
			<td>HITACHI</td>
			<td></td>
			<td>T15A43</td>
		</tr>
		<tr>
			<td>SDS-PAGE running buffer</td>
			<td></td>
			<td></td>
			<td>25 mM Tris/ 192 mM Glycine/ 0.1% SDS</td>
		</tr>
		<tr>
			<td>SDS-PAGE sample buffer (2x)</td>
			<td></td>
			<td></td>
			<td>0.1M Tris-HCl (pH 6.8)/ 4% SDS/ 20% glycerol/ 0.01% BPB/12% 2-merpaptoethanol</td>
		</tr>
		<tr>
			<td>SDS-polyacrylamide gel</td>
			<td></td>
			<td></td>
			<td>4-20% gradient polyacrylamide gel</td>
		</tr>
		<tr>
			<td>Skim milk powder</td>
			<td>FUJIFILM Wako Pure Chemical</td>
			<td>190-12865</td>
			<td>For Western blotting blocking buffer (5% skim milk/ 0.1% Tween 20/ PBS)</td>
		</tr>
		<tr>
			<td>Total cholesterol assay kit</td>
			<td>FUJIFILM Wako Pure Chemical</td>
			<td>439-17501</td>
			<td>For lipids measurment</td>
		</tr>
		<tr>
			<td>Triglyicerides assay kit</td>
			<td>FUJIFILM Wako Pure Chemical</td>
			<td>432-40201</td>
			<td>For lipids measurment</td>
		</tr>
		<tr>
			<td>Tube slicer for thick-walled tube</td>
			<td>BECKMAN COULTER</td>
			<td>347960</td>
			<td>For lipoprotein isolation</td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>SIGMA-ALDRICH</td>
			<td>P1379</td>
			<td>For Western blotting washing buffer (0.1% Tween 20/ PBS)</td>
		</tr>
		<tr>
			<td>Ultracentrifuge</td>
			<td>BECKMAN COULTER</td>
			<td>A95761</td>
			<td>Optima MAX-TL</td>
		</tr>
		<tr>
			<td>Western blotting wet transfer system</td>
			<td>BIO-RAD</td>
			<td></td>
			<td>Mini Trans-Blot Cell</td>
		</tr>
	</tbody>
</table>

isolation and analysis of plasma lipoproteins by ultracentrifugation

血浆脂蛋白和非政治蛋白的分析是血脂血症诊断和脂质代谢和动脉粥样硬化研究的重要组成部分。虽然有几种方法分析等离子脂蛋白，超中心化仍然是最流行和最可靠的方法之一。由于其完整的分离程序，通过这种方法分离的脂蛋白分数可用于分析脂蛋白，阿波利波蛋白，蛋白质组，和功能研究的脂蛋白与培养细胞体外。在这里，我们提供了隔离七个脂蛋白分数的详细协议，包括 VLDL（d=lt;1.006 g/mL）、IDL（d=1.02 g/mL）、LDL（d=1.04 和 1.06 g/mL）、硬盘（d=1.08， 1.10 和 1.21 克/mL） 从兔子血浆使用顺序浮动超中心。此外，我们向读者介绍如何分析阿波利波蛋白，如apoA-I，apoB，和apoE由SDS-PAGE和西方印迹，并显示具有代表性的结果脂蛋白和非波利波蛋白配置文件使用高脂兔模型。这种方法可以成为临床医生和基础科学家分析脂蛋白功能的标准方案。

使用此协议，我们使用 1 mL 的血浆分离兔子脂蛋白，并获得了 7 个密度分数。对于大多数研究目的，孤立的密度分数足以测量上文所述的脂质和非政治蛋白。同样的程序也可用于将等离子脂蛋白与人类和其他物种分离。对于小动物，如小鼠，需要汇集血浆。图 3显示了野生类型 （WT） 兔子喂养正常标准 （NS） 饮食或高胆固醇 （HC） 饮食的脂蛋白特征。兔子是食草动物，因?...

Watch this Scientific Journal Video about Isolation and Analysis of Plasma Lipoproteins by Ultracentrifugation at JoVE.com

Isolation and Analysis of Plasma Lipoproteins by Ultracentrifugation

用于分析等离子脂蛋白的几种方法：然而，超中心化仍然是最流行和最可靠的方法之一。在这里，我们描述了如何使用顺序密度超中心化将脂蛋白从血浆中分离的方法，以及如何分析用于诊断和研究目的的非脊髓灰质素。

超中心对等离子脂蛋白的分离和分析

Analysis of plasma lipoproteins and apolipoproteins is an essential part for the diagnosis of dyslipidemia and studies of lipid metabolism and ...

血浆脂蛋白的分析对于高脂血症的诊断和动脉粥样硬化研究具有十分重要的意义。在此协议中，我们将引入使用超中心化分离等离子脂蛋白的基本方法。这种方法允许研究人员使用一毫升血浆分离七个脂蛋白分数。这些分数可用于脂质、阿波利波蛋白以及功能良好的研究这种方法可用于人类和动物样本，用于诊断血脂异常和相关疾病。首先将一毫升等离子体转移到每个聚碳酸酯超中分子管中。然后将管子装在固定角度转子中，在 35.6 万倍 G 下将等离子体离心，在 4 摄氏度下进行 2.5 小时。将管切片机中刀片的位置调整到顶部部分和底部部分之间的水平。使用切片机切割管子，并将顶部部分（约 200 微升）收集到新的微管中。将剩余的底部部分收集到一个新的聚碳酸酯超中分子管中，用于下一次分离。通过添加相同的密度解决方案，将总体积调整为 800 微升。通过添加 58.9 微升每毫升溶液 1.21 克的密度，将底部分数调整为每毫升 1.02 克的密度，为每毫升溶液添加 141.1 微升，总体积为 1 毫升。将这些管子装在固定角转子和离心机中，在 356，000 倍 G 中加载 2.5 小时，温度为 4 摄氏度。继续收集最高分数，调整底部分数的密度，离心，每毫升的分数密度为 1.02、1.04、1.06、1.08、1.1 和 1.21 克。对于最后的超中心，离心机在51.3万倍G四个小时在4摄氏度。这里显示了喂食正常标准饮食或高胆固醇饮食的兔子的脂蛋白特征。兔子是食草动物，因此它们的血浆TC、TG和PL水平通常低于人类和小鼠。在正常标准饮食喂养的兔子中，TC主要分布在HDL3和低密度脂蛋白中。在正常标准饮食的野生类型兔子中，39%的血浆TG分布在VLDL中，而57%的血浆PL包含在HDL3中。当兔子受到高胆固醇饮食的挑战时，它们会迅速发展成高胆固醇血症。他们的脂蛋白特征是VLDLs的显著升高。 野生型兔子的七个脂蛋白分数以正常标准或高胆固醇饮食为食，在SDS聚丙烯酰胺凝胶上运行，并带有CBB染色。在高胆固醇饮食中，VLDL、IDL 和 LDL 中的 ApoB-100 和 ApoE 含量均显著升高。西方的印迹被用来比较三种不同高脂兔子的无脊髓灰质素和脂蛋白特征：高胆固醇饮食喂养的野生类型兔子、ApoE淘汰兔子和患有低密度脂蛋白受体缺陷的渡边可遗传超脂兔子。含有高胆固醇饮食喂养野生类型兔子颗粒的ApoB的特点是增加ApoB-100和ApoE含量，而ApoE淘汰兔子显示ApoB-48随着ApoA-1的出现而增加。渡边可遗传性高脂兔子在基因上缺乏低密度脂蛋白受体功能，因此在LDL中ApoB-100显著增加，在高密度脂蛋白中发现ApoA-1减少，类似于人类家族性高胆固醇血症患者。在此过程中，样本恢复是最关键的。为了尽量减少样品损失，应小心收集分数，并完全溶解底部部分的粘性沉淀。收集七个脂蛋白分数后，需要透析进行排序。透析完成后，脂蛋白样品使用SDS页面、西式印迹和基于细胞的研究进行分析。在临床环境中，超中心化通常将脂蛋白分成四个部分。目前的方法可以分离七个脂蛋白分数，这提供了脂蛋白的更多细节，用于研究高脂血症。

Research

JoVE Journal

Biochemistry

Isolation of High-density Lipoproteins for Non-coding Small RNA Quantification

高密度脂蛋白的分离的非编码小RNA定量

The diversity of small non-coding RNAs (sRNA) is rapidly expanding and their roles in biological processes, including gene regulation, are emerging. Most ...

科研

生物化学

Lipid Droplet Isolation for Quantitative Mass Spectrometry Analysis

脂滴隔离定量质谱分析

Lipid droplets are vital to the replication of a variety of different pathogens, most prominently the Hepatitis C Virus (HCV), as the putative site of ...

Protein Digestion, Ultrafiltration, and Size Exclusion Chromatography to Optimize the Isolation of Exosomes from Human Blood Plasma and Serum

蛋白质消化、超滤和尺寸排除色谱法优选人血浆和血清外来体的分离

Exosomes, a type of nanovesicle released from all cell types, can be isolated from any bodily fluid. The contents of exosomes, including proteins and ...

Measuring Trans-Plasma Membrane Electron Transport by C2C12 Myotubes

C2C12 Myotubes 测量反式等离子体膜电子传输

Trans-plasma membrane electron transport (tPMET) plays a role in protection of cells from intracellular reductive stress as well as protection from damage ...

High-Resolution Complexome Profiling by Cryoslicing BN-MS Analysis

通过低温BN-MS分析进行高分辨率复杂分析

Proteins generally exert biological functions through interactions with other proteins, either in dynamic protein assemblies or as a part of stably formed ...

Ubiquitin Chain Analysis by Parallel Reaction Monitoring

通过并行反应监测进行无处不在的链分析

Assessment of the global profile of ubiquitin chain topologies within a proteome is of interest to answer a wide range of biological questions. The ...

Single Cell Analysis Of Transcriptionally Active Alleles By Single Molecule FISH

单分子鱼的转录活性等位基因单细胞分析

Gene transcription is an essential process in cell biology, and allows cells to interpret and respond to internal and external cues. Traditional bulk ...

Semi-Quantitative Analysis of Peptidoglycan by Liquid Chromatography Mass Spectrometry and Bioinformatics

肽聚糖的液相色谱质谱和生物信息学半定量分析

Peptidoglycan is an important component of bacterial cell walls and a common cellular target for antimicrobials. Although aspects of peptidoglycan ...

Spot Variation Fluorescence Correlation Spectroscopy for Analysis of Molecular Diffusion at the Plasma Membrane of Living Cells

点变异荧光相关光谱用于分析活细胞质膜上的分子扩散

Dynamic biological processes in living cells, including those associated with plasma membrane organization, occur on various spatial and temporal scales, ...

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 ...