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

This work was supported in part by a research grants from JSPS KAKENHI Grant Number JP 20K08858, the National Natural Science Foundation of China (No. 81941001 and 81770457), JSPS-CAS under the Japan-China Research Cooperative Program.

All procedures for rabbit studies were performed with approval of University of Yamanashi Institutional Animal Care and Use Committee (Approved number: A28-39).
1. Plasma separation from rabbit blood
<ol>
	<li>Prepare 1.5 mL microtubes containing 15 &#181;L of 0.5 M EDTA (pH 8.0) for blood collection.</li>
	<li>Put a rabbit in a restrainer and puncture an auricular intermediate artery using a 22 gauge needle and collect blood into a tube. Mix the blood with EDTA gently and put them on ice.</...

<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>

Hyperlipidemia is one of the most important risk factors of atherosclerotic disease. Thus, analysis of plasma lipoproteins is not only essential for diagnosis of dyslipidemia patients but also important for investigation of molecular mechanisms of lipoprotein metabolism and atherosclerosis. In this study, we described the protocol of isolation and analysis of plasma lipoproteins which can be applied in the laboratories where ultracentrifugation is available. Information obtained by this method is comprehensive and straig...

Dyslipidemia is the major risk factor of atherosclerotic disease in the world. High levels of low-density lipoproteins (LDLs) and low levels of high-density lipoproteins (HDLs) are closely associated with a high risk of coronary heart disease (CHD)1,2. In the clinical setting, both LDL-cholesterol (LDL-C) and HDL-cholesterol (HDL-C) are routinely measured using an automated analyzer in a clinical laboratory3,4. Despite this, it is essential to analyze lipoprotein profiles in details for the diagnosis of dyslipidemia and the study of lipid metabolism and atherosclerosis in human and experimental animals. Several methods have been reported to analyze plasma lipoproteins such as ultracentrifugation, size exclusion chromatography [fast protein liquid chromatography (FPLC) and high performance liquid chromatography (HPLC)], electrophoresis by agarose and polyacrylamide gels, nuclear magnetic resonance, and selective chemical precipitation using polyanions and divalent cations or other chemicals. In 1950s, Havel&#8217;s group first proposed the concept of lipoproteins defined by densities using ultracentrifugation and classified them into chylomicrons (CM), very-low-density lipoproteins (VLDL), intermediate-density lipoproteins (IDL), LDL, and HDL5 and later on, the method was further modified by other groups6,7. Until now, ultracentrifugation is the most popular and reliable method while the practical protocol is still not available. In this paper, we attempted to describe an easy-to-use protocol for isolating a small scale of plasma using sequential density floating ultracentrifugation originally described previously8. Isolation of seven plasma lipoprotein fractions [VLDL (d&#60;1.006 g/mL), IDL (d=1.02 g/mL), LDLs (d=1.04 and 1.06 g/mL), HDLs (d=1.08, 1.10, and 1.21 g/mL)] enables researchers to make an extensive analysis of both lipoproteins and their compositional apolipoproteins9,10,11. The intact seven consecutive density lipoproteins can be used for analyzing lipoprotein functions and also serving to&#160;cell-based in vitro strategies. This protocol should be useful for both clinical diagnosis and basic research. Here we used&#160;rabbit plasma as an example to demonstrate this technique while plasma from other species can be applied in the same way.

<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

Analysis of plasma lipoproteins and apolipoproteins is an essential part for the diagnosis of dyslipidemia and studies of lipid metabolism and atherosclerosis. Although there are several methods for analyzing plasma lipoproteins, ultracentrifugation is still one of the most popular and reliable methods. Because of its intact separation procedure, the lipoprotein fractions isolated by this method can be used for analysis of lipoproteins, apolipoproteins, proteomes, and functional study of lipoproteins with cultured cells in vitro. Here, we provide a detailed protocol to isolate seven lipoprotein fractions including VLDL (d&#60;1.006 g/mL), IDL (d=1.02 g/mL), LDLs (d=1.04 and 1.06 g/mL), HDLs (d=1.08, 1.10, and 1.21 g/mL) from rabbit plasma using sequential floating ultracentrifugation. In addition, we introduce the readers how to analyze apolipoproteins such as apoA-I, apoB, and apoE by SDS-PAGE and Western blotting and show representative results of lipoprotein and apolipoprotein profiles using hyperlipidemic rabbit models. This method can become a standard protocol for both clinicians and basic scientists to analyze lipoprotein functions.

Using this protocol, we isolated rabbit lipoproteins using 1 mL of plasma and obtained seven density fractions. Isolated density fractions are enough for measuring lipids and apolipoproteins as described above for most research purposes. The same procedure can also be used for isolating plasma lipoproteins from human and other species. For small-sized animals such as mice, pooled plasma is required. Figure 3 shows lipoprotein profiles of wild-type (WT) rabbits fed either a normal standard (N...

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

Several methods have been used for analyzing plasma lipoproteins; however, ultracentrifugation is still one of the most popular and reliable methods. Here, we describe a method regarding how to isolate lipoproteins from plasma using sequential density ultracentrifugation and how to analyze the apolipoproteins for both diagnostic and research purposes.

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

Research

JoVE Journal

Biochemistry

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

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

Lipid Droplet Isolation for Quantitative Mass Spectrometry Analysis

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

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

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

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

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

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

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

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

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