This study was supported by the National Research Foundation of Korea, Grant NRF-2018R1D1A1A09084044. J. H. Lee was supported by a research grant from Kwangwoon University in 2019. Hyerin Kim was supported by the &#8220;Competency Development Program for Industry Specialists&#8221; of the Korean Ministry of Trade, Industry and Energy (MOTIE), operated by the Korea Institute for Advancement of Technology (KIAT) (No. P0002397, HRD program for Industrial Convergence of Wearable Smart Devices).

1. Device fabrication
<ol>
	<li>Define the region to be printed on paper using printer software (Table of Materials). 
	NOTE: The design has 12 wax-patterned layers in which the diameters of the circular sample areas gradually narrow from 5 mm to 2 mm (Figure 1A).</li>
	<li>Print hydrophobic wax on the designated regions on both sides of the cellulose paper (Table of Materials) using a commercial wax printer (Table of Materials) (Figure 1B).</li>
	<li>Place the wax-printed paper in a laboratory oven for 80 s at 120 &#176;C. 
	NOTE: This step allows the wax to reach the inside of the paper by achieving a thermal reflow of the printed wax. With this incubation protocol, the resolution of the pattern is ~2 mm. Thus, be careful not to print patterns of less than 2 mm. Otherwise the patterns will be blocked by the wax.</li>
	<li>Cut the wax-printed paper with a cutter to make individual devices (Figure 1C).</li>
	<li>Drop 5 &#956;L and 2 &#956;L of permselective membrane (e.g., Nafion; Table of Materials) onto the sample areas in the leftmost and rightmost layers, respectively (Figure 1D).</li>
	<li>Place the layers coated with the permselective membrane on a hot plate at 70 &#176;C for 30 min to evaporate the permselective membrane solvent.</li>
	<li>Seal the outermost surface of the coated layer facing the buffer solution with pressure-sensitive tape, leaving a small hole.</li>
	<li>Fold the printed individual device (i.e., Exo-PAD) back and forth along the white lines. 
	NOTE: By folding the device, all sample areas become convergently connected (Figure 1E). This convergent design focuses the electric field lines when the voltage is applied for ICP, achieving more intensive preconcentration of the microvesicles and exosomes.</li>
</ol>
2. Enrichment and spatial focusing of microvesicles and exosomes by ion concentration polarization
<ol>
	<li>Load 15 &#956;L of the microvesicle and exosome sample (~3 x 1011 particles/mL in 0.1x phosphate buffered saline [PBS] with 0.05% Tween 20) in the convergent sample areas by pipetting and wait a few seconds to ensure complete wetting of all sample areas (Figure 1F).</li>
	<li>Place two acrylic chambers at both ends of the Exo-PAD and clamp the Exo-PAD securely with small binder clips to prevent unfolding (Figure 1G).</li>
	<li>Fill the chambers with 110 &#956;L of 0.1x PBS and insert two Ag/AgCl electrodes (Figure 1H).</li>
	<li>Apply 30 V to the electrodes for 20 min using a current-voltage source measurement system (Table of Materials). 
	NOTE: The applied voltage generates the ICP phenomenon and hence preconcentrates the microvesicles and exosomes on layers 8 and 9 of the Exo-PAD.</li>
</ol>
3. Isolation of the enriched microvesicles and exosomes
<ol>
	<li>Separate the folded Exo-PAD from the acrylic chambers and unfold the device to isolate the enriched microvesicles and exosomes from the other layers (Figure 1I).</li>
	<li>Punch out the sample areas in layers 8 and 9, where the microvesicles and exosomes are enriched, for downstream analysis (Figure 1J).</li>
</ol>
4. Scanning electron microscopy analysis
<ol>
	<li>Fix the enriched microvesicles and exosomes by immersing the punched areas in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer for 1 h and in 1% osmium tetroxide in 0.1 M sodium cacodylate for 1 h. 
	CAUTION: Osmium tetroxide is highly poisonous and hazardous chemical. Because osmium tetroxide can penetrate plastics, it must be stored in glass. Any handling of osmium tetroxide must be performed in a chemical fume hood with double nitrile gloves.</li>
	<li>Dehydrate the fixed microvesicles and exosomes with ascending grades of 200 proof ethanol (i.e., 50%, 70%, 90%, and 100%) for 30 min each.</li>
	<li>Chemically dry the sample by immersing it in hexamethyldisilazane in a desiccator for 30 min. 
	CAUTION: Hexamethyldisilazane is a flammable and moisture-sensitive chemical. It must be stored in a dry and well-ventilated area away from ignition sources.</li>
	<li>Coat the completely dried microvesicles and exosomes with gold/palladium (~20 nm) via sputtering and capture scanning electron microscopy (SEM) images.</li>
</ol>
5. Nanoparticle tracking analysis
<ol>
	<li>Punch out the sample areas in layers 6, 8, and 10 using a biopsy punch after 20 min of device operation.</li>
	<li>Immerse each punched-out area in buffer solution (0.1x PBS with 0.01% Tween 20).</li>
	<li>Vortex for 10 min and centrifuge for 30 s at 6,000 rpm to resuspend the enriched microvesicles and exosomes.</li>
	<li>Remove the punched-out areas from the solution and measure the concentration of microvesicles and exosomes by a nanoparticle tracking analysis (NTA) instrument (Table of Materials).</li>
</ol>

<ol>
	<li>Edgar, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Q+&amp;+A:+What+are+exosomes,+exactly.">Q &amp; A: What are exosomes, exactly.</a> BMC Biology. 14 (1), 1-7 (2016).</li><li>Contreras-Naranjo, J. C., Wu, H. J., Ugaz, V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidics+for+exosome+isolation+and+analysis:+Enabling+liquid+biopsy+for+personalized+medicine.">Microfluidics for exosome isolation and analysis: Enabling liquid biopsy for personalized medicine.</a> Lab on a Chip. 17 (21), 3558-3577 (2017).</li><li>Simons, M., Raposo, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exosomes+-+vesicular+carriers+for+intercellular+communication.">Exosomes - vesicular carriers for intercellular communication.</a> Current Opinion in Cell Biology. 21 (4), 575-581 (2009).</li><li>St&#229;hl, A. L., Johansson, K., Mossberg, M., Kahn, R., Karpman, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exosomes+and+microvesicles+in+normal+physiology,+pathophysiology,+and+renal+diseases.">Exosomes and microvesicles in normal physiology, pathophysiology, and renal diseases.</a> Pediatric Nephrology. 34 (1), 11-30 (2019).</li><li>Chen, C., Lin, B. R., Hsu, M. Y., Cheng, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paper-based+devices+for+isolation+and+characterization+of+extracellular+vesicles.">Paper-based devices for isolation and characterization of extracellular vesicles.</a> Journal of Visualized Experiments. (98), e52722 (2015).</li><li>Kim, 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=Origami-paper-based+device+for+microvesicle/exosome+preconcentration+and+isolation.">Origami-paper-based device for microvesicle/exosome preconcentration and isolation.</a> Lab on a Chip. 19 (23), 3917-3921 (2019).</li><li>Raposo, G., Stoorvogel, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+vesicles:+Exosomes,+microvesicles,+and+friends.">Extracellular vesicles: Exosomes, microvesicles, and friends.</a> Journal of Cell Biology. 200 (4), 373-383 (2013).</li><li>Lee, Y., El Andaloussi, S., Wood, M. J. 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The authors have nothing to disclose.

Although the Exo-PAD was used successfully for the enrichment and isolation of microvesicles and exosomes, several critical points should be carefully considered: 1) the oven incubation time and temperature during the device preparation, 2) processing time, 3) application of voltage with varying layer numbers and sample area diameters, and 4) applicability to clinical samples.
The incubation time and temperature given in the protocol are optimized conditions to fabricate a reliable device. Lon...

Microvesicles and exosomes are small membrane vesicles measuring 0.2&#8722;1 &#956;m and 30&#8722;200 nm, respectively. They are secreted into the extracellular environment by several different cell types1,2,3,4,5. They contain parental cell information in the form of subsets of DNA, mRNA, miRNA, proteins, and lipids, and circulate throughout the body via various body fluids such as serum, plasma, urine, cerebrospinal fluid, amniotic fluid, and saliva6,7,8,9. Thus, techniques for efficient isolation of microvesicles and exosomes from biological fluids can provide extensive opportunities in the fields of the diagnosis, prognosis, and real-time monitoring of disease, as well as in the development of new therapeutics.
However, the conventional isolation method for microvesicles and exosomes based on ultracentrifugation is extremely time-consuming and causes significant loss and contamination of the sample. This is because it involves several cumbersome pipetting and loading steps and discarding of various reagents with repeated ultracentrifugation5,6,10,11,12. Moreover, the high shear stress induced by ultracentrifugation (~100,000 x g) can cause the physical lysis of microvesicles and exosomes, yielding poor recovery rates (5&#8722;23%)6,13,14. Therefore, a highly efficient, unobtrusive isolation technique for microvesicles and exosomes must be developed to reduce damage and loss, thereby achieving higher recovery rates.
An origami-paper-based device (Exo-PAD) was developed for simpler, gentler, and highly efficient isolation of microvesicles and exosomes6. The design of the Exo-PAD is a multifolded paper with serially connected sample areas that gradually decrease in diameter. The ion concentration polarization (ICP) technique, which is a nano-electrokinetic phenomenon that preconcentrates charged biomolecules, was integrated with this unique design. Using the Exo-PAD resulted in fivefold enrichment of the microvesicles and exosomes in specific layers and their isolation by simply unfolding the device. This article describes the Exo-PAD in detail, from the overall device fabrication and operation to analysis of its use, to illustrate the method and show representative results6.

<table border="0" cellpadding="0" cellspacing="0" style="width:1356px;" width="1355">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:397px;">Ag/AgCl electrodes</td>
			<td style="width:207px;">A-M Systems, Inc.</td>
			<td style="width:119px;">531500</td>
			<td style="width:633px;">0.15&#34; diameter</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Albumin from Bovine Serum (BSA), Alexa Fluor 594 conjugate</td>
			<td>Thermo Fisher Scientific</td>
			<td>A13101</td>
			<td>BSA conjugated with Alexa Fluor 594 (Ex/Em: 590/617 nm)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Carbonate-Bicarbonate Buffer</td>
			<td>Sigma-Aldrich</td>
			<td>C3041-50CAP</td>
			<td>Carbonate buffer</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CorelDraw software (Coral Co., Canada)</td>
			<td>Corel Corporation</td>
			<td></td>
			<td>Printer software to define wax printing region</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ColorQube 8870</td>
			<td>Xerox Corporation</td>
			<td></td>
			<td>Wax printer</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Chromatography paper grade 1</td>
			<td>Whatman</td>
			<td>3001-861</td>
			<td>Cellulose paper, dimension: 20 * 20 cm</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fluorescent-labeled exosome standards</td>
			<td>HansaBioMed Life Sciences, Ltd.</td>
			<td>HBM-F-PEP-100</td>
			<td>Exosome labeled with FITC (Ex/Em: 490/520 nm)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Keithley 2410 current/voltage source-meter</td>
			<td>Keithley Instruments, Inc.</td>
			<td style="width:119px;"></td>
			<td>Current&#8211;voltage source measurement system</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Nafion perfluorinated resin solution</td>
			<td>Sigma-Aldrich</td>
			<td>31175-20-9</td>
			<td>Permselective membrane, 20 wt.% in the mixture of lower aliphatic alcohols and water; contains 34% water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NanoSight LM10</td>
			<td>NanoSight Technology</td>
			<td style="width:119px;"></td>
			<td>Nanoparticle tracking analysis (NTA) machine</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phosphate-buffered saline (PBS, pH7.4)</td>
			<td>Thermo Fisher Scientific</td>
			<td>10010001</td>
			<td></td>
		</tr>
	</tbody>
</table>

paper-based preconcentration and isolation of microvesicles and exosomes

Microvesicles and exosomes are small membranous vesicles released to the extracellular environment and circulated throughout the body. Because they contain various parental cell-derived biomolecules such as DNA, mRNA, miRNA, proteins, and lipids, their enrichment and isolation are critical steps for their exploitation as potential biomarkers for clinical applications. However, conventional isolation methods (e.g., ultracentrifugation) cause significant loss and damage to microvesicles and exosomes. These methods also require multiple repetitive steps &#160;of ultracentrifugation, loading, and wasting of reagents. This article describes a detailed method to fabricate an origami-paper-based device (Exo-PAD) designed for the effective enrichment and isolation of microvesicles and exosomes in a simple manner. The unique design of the Exo-PAD, consisting of accordion-like multifolded layers with convergent sample areas, is integrated with the ion concentration polarization technique, thereby enabling fivefold enrichment of the microvesicles and exosomes on specific layers. In addition, the enriched microvesicles and exosomes are isolated by simply unfolding the Exo-PAD.

The operation time must be optimized to achieve the maximum recovery yield of the enriched microvesicles and exosomes. Insufficient time does not allow sufficient migration of the microvesicles and exosomes, which decreases the enrichment, whereas excessive time deteriorates the spatial focusing and hence disperses the microvesicles and exosomes. Thus, through the time optimization step, the maximum preconcentration factor of microvesicles and exosomes and the final location where microvesicles and exosomes are most enri...

Watch this Scientific Journal Video about Paper-Based Preconcentration and Isolation of Microvesicles and Exosomes at JoVE.com

Paper-Based Preconcentration and Isolation of Microvesicles and Exosomes

Presented is a protocol to fabricate a paper-based device for the effective enrichment and isolation of microvesicles and exosomes.

Microvesicles and exosomes are small membranous vesicles released to the extracellular environment and circulated throughout the body. Because they ...

paper-based-preconcentration-isolation-microvesicles

Universidad de Cartagena UDEC

Research

JoVE Journal

Biochemistry

6.0K Views.  Kwangwoon University. Presented is a protocol to fabricate a paper-based device for the effective enrichment and isolation of microvesicles and exosomes.

Video: Paper-Based Preconcentration and Isolation of Microvesicles and Exosomes

Isolation and Respiratory Measurements of Mitochondria from Arabidopsis thaliana

Mitochondria are essential organelles involved in numerous metabolic pathways in plants, most notably the production of adenosine triphosphate (ATP) from the oxidation of reduced compounds such as nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FADH2). The complete annotation of the Arabidopsis thaliana genome has established it as the most widely used plant model system, and thus the need to purify mitochondria from a variety of organs (leaf, root, or flower) is necessary to fully utilize the tools that are now available for Arabidopsis to study mitochondrial biology. Mitochondria are isolated by homogenization of the tissue using a variety of approaches, followed by a series of differential centrifugation steps producing a crude mitochondrial pellet that is further purified using continuous colloidal density gradient centrifugation. The colloidal density material is subsequently removed by multiple centrifugation steps. Starting from 100 g of fresh leaf tissue, 2 - 3 mg of mitochondria can be routinely obtained. Respiratory experiments on these mitochondria display typical rates of 100 - 250 nmol O2 min-1 mg total mitochondrial protein-1 (NADH-dependent rate) with the ability to use various substrates and inhibitors to determine which substrates are being oxidized and the capacity of the alternative and cytochrome terminal oxidases. This protocol describes an isolation method of mitochondria from Arabidopsis thaliana leaves using continuous colloidal density gradients and an efficient respiratory measurements of purified plant mitochondria.

Isolation and Respiratory Measurements of Mitochondria from Arabidopsis thaliana

As mitochondria are only a small percentage of the plant cell, they need to be purified for a range of studies. Mitochondria can be isolated from a variety of plant organs by homogenization, followed by differential and density gradient centrifugation to obtain a highly purified mitochondrial fraction.

Mitochondria are essential organelles involved in numerous metabolic pathways in plants, most notably the production of adenosine triphosphate (ATP) from ...

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 RNAs, are unique to the cells from which they are derived and can be used as indicators of disease. Several common enrichment protocols, including ultracentrifugation, yield exosomes laden with soluble protein contaminants. Specifically, we have found that the most abundant proteins within blood often co-purify with exosomes and can confound downstream proteomic studies, thwarting the identification of low abundance biomarker candidates. Of additional concern is irreproducibility of exosome protein quantification due to inconsistent representation of non-exosomal protein levels. The protocol detailed here was developed to remove non-exosomal proteins that co-purify along with exosomes, adding rigor to the exosome purification process. Five methods were compared using paired blood plasma and serum from five donors. Analysis using nanoparticle tracking analysis and micro bicinchoninic acid protein assay revealed that a combined protocol utilizing ultrafiltration and size exclusion chromatography yielded the optimal vesicle enrichment and soluble protein removal. Western blotting was used to verify that the expected abundant blood proteins, including albumin and apolipoproteins, were depleted.

Here, we present a protocol to purify exosomes from both plasma and serum with reduced co-purification of non-exosomal blood proteins. The optimized protocol includes ultrafiltration, protease treatment, and size exclusion chromatography. Enhanced purification of exosomes benefits downstream analyses, including more accurate quantification of vesicles and proteomic characterization.

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

Rapid Isolation of the Mitoribosome from HEK Cells

The human mitochondria possess a dedicated set of ribosomes (mitoribosomes) that translate 13 essential protein components of the oxidative phosphorylation complexes encoded by the mitochondrial genome. Since all proteins synthesized by human mitoribosomes are integral membrane proteins, human mitoribosomes are tethered to the mitochondrial inner membrane during translation. Compared to the cytosolic ribosome the mitoribosome has a sedimentation coefficient of 55S, half the rRNA content, no 5S rRNA and 36 additional proteins. Therefore, a higher protein-to-RNA ratio and an atypical structure make the human mitoribosome substantially distinct from its cytosolic counterpart.
Despite the central importance of the mitoribosome to life, no protocols were available to purify the intact complex from human cell lines. Traditionally, mitoribosomes were isolated from mitochondria-rich animal tissues that required kilograms of starting material. We reasoned that mitochondria in dividing HEK293-derived human cells grown in nutrient-rich expression medium would have an active mitochondrial translation, and, therefore, could be a suitable source of material for the structural and biochemical studies of the mitoribosome. To investigate its structure, we developed a protocol for large-scale purification of intact mitoribosomes from HEK cells. Herein, we introduce nitrogen cavitation method as a faster, less labor-intensive and more efficient alternative to traditional mechanical shear-based methods for cell lysis. This resulted in preparations of the mitoribosome that allowed for its structural determination to high resolution, revealing the composition of the intact human mitoribosome and its assembly intermediates. Here, we follow up on this work and present an optimized and more cost-effective method requiring only ~1010 cultured HEK cells. The method can be employed to purify human mitoribosomal translating complexes, mutants, quality control assemblies and mitoribosomal subunits intermediates. The purification can be linearly scaled up tenfold if needed, and also applied to other types of cells.

Mitochondria have specialized ribosomes that diverged from their bacterial and cytoplasmic counterparts. Here we show how mitoribosomes can be obtained from their native compartment in HEK cells. The method involves isolation of mitochondria from suspension cells and consequent purification of mitoribosomes.

The human mitochondria possess a dedicated set of ribosomes (mitoribosomes) that translate 13 essential protein components of the oxidative ...

Detection and Isolation of Apoptotic Bodies to High Purity

Apoptotic bodies (ApoBDs), microvesicles and exosomes are the key members of the extracellular vesicle family, with ApoBDs being one of the largest type. It has been proposed that ApoBDs can aid cell clearance as well as intercellular communication through trafficking biomolecules. Conventional approaches used for the identification and isolation of ApoBDs are often limited by the lack of accurate quantification and low sample purity. Here, we describe a workflow to confirm the induction of apoptosis, validate ApoBD formation, and isolate ApoBDs to high purity. We will also outline and compare fluorescence-activated cell sorting (FACS) and differential centrifugation based approaches to isolate ApoBDs. Furthermore, the purity of isolated ApoBDs will be confirmed using a previously establish flow cytometry-based staining and analytical method. Taken together, using the described approach, THP-1 monocyte apoptosis and apoptotic cell disassembly was induced and validated, and ApoBD generated from THP-1 monocytes were isolated to a purity of 97-99%.

A workflow using flow cytometry or differential centrifugation is developed to detect, quantify and isolate apoptotic bodies from an apoptotic sample to high purity.

Apoptotic bodies (ApoBDs), microvesicles and exosomes are the key members of the extracellular vesicle family, with ApoBDs being one of the largest type. ...

Isolation of Physiologically Active Thylakoids and Their Use in Energy-Dependent Protein Transport Assays

Chloroplasts are the organelles in green plants responsible for carrying out numerous essential metabolic pathways, most notably photosynthesis. Within the chloroplasts, the thylakoid membrane system houses all the photosynthetic pigments, reaction center complexes, and most of the electron carriers, and is responsible for light-dependent ATP synthesis. Over 90% of chloroplast proteins are encoded in the nucleus, translated in the cytosol, and subsequently imported into the chloroplast. Further protein transport into or across the thylakoid membrane utilizes one of four translocation pathways. Here, we describe a high-yield method for isolation of transport-competent thylakoids from peas (Pisum sativum), along with transport assays through the three energy-dependent cpTat, cpSec1, and cpSRP-mediated pathways. These methods enable experiments relating to thylakoid protein localization, transport energetics, and the mechanisms of protein translocation across biological membranes.

We present protocols herein for high-yield isolation of physiologically active thylakoids and protein transport assays for the chloroplast twin arginine translocation (cpTat), secretory (cpSec1), and signal recognition particle (cpSRP) pathways.

Chloroplasts are the organelles in green plants responsible for carrying out numerous essential metabolic pathways, most notably photosynthesis. Within ...

Isolation of Glomeruli and In Vivo Labeling of Glomerular Cell Surface Proteins

Proteinuria results from the disruption of the glomerular filter that is composed of the fenestrated endothelium, glomerular basement membrane, and podocytes with their slit diaphragms. The delicate structure of the glomerular filter, especially the slit diaphragm, relies on the interplay of diverse cell surface proteins. Studying these cell surface proteins has so far been limited to in vitro studies or histologic analysis. Here, we present a murine in vivo biotinylation labeling method, which enables the study of glomerular cell surface proteins under physiologic and pathophysiologic conditions. This protocol contains information on how to perfuse mouse kidneys, isolate glomeruli, and perform endogenous immunoprecipitation of a protein of interest. Semi-quantitation of glomerular cell surface abundance is readily available with this novel method, and all proteins accessible to biotin perfusion and immunoprecipitation can be studied. In addition, isolation of glomeruli with or without biotinylation enables further analysis of glomerular RNA and protein as well as primary glomerular cell culture (i.e., primary podocyte cell culture).

Isolation of Glomeruli and In Vivo Labeling of Glomerular Cell Surface Proteins

Here we present a protocol for murine in vivo labeling of glomerular cell surface proteins with biotin. This protocol contains information on how to perfuse mouse kidneys, isolate glomeruli, and perform endogenous immunoprecipitation of the protein of interest.

Proteinuria results from the disruption of the glomerular filter that is composed of the fenestrated endothelium, glomerular basement membrane, and ...

Looking Outwards: Isolation of Cyanobacterial Released Carbohydrate Polymers and Proteins

Cyanobacteria can actively secrete a wide range of biomolecules into the extracellular environment, such as heteropolysaccharides and proteins. The identification and characterization of these biomolecules can improve knowledge about their secretion pathways and help to manipulate them. Furthermore, some of these biomolecules are also interesting in terms of biotechnological applications. Described here are two protocols for easy and rapid isolation of cyanobacterial released carbohydrate polymers and proteins. The method for isolation of released carbohydrate polymers is based on conventional precipitation techniques of polysaccharides in aqueous solutions using organic solvents. This method preserves the characteristics of the polymer and simultaneously avoids the presence of contaminants from cell debris and culture medium. At the end of the process, the lyophilized polymer is ready to be used or characterized or can be subjected to further rounds of purification, depending on the final intended use. Regarding the isolation of the cyanobacterial exoproteome, the technique is based on the concentration of the cell-free medium after removal of the major contaminants by centrifugation and filtration. This strategy allows for reliable isolation of proteins that reach the extracellular milieu via membrane transporters or outer membrane vesicles. These proteins can be subsequently identified using standard mass spectrometry techniques. The protocols presented here can be applied not only to a wide range of cyanobacteria, but also to other bacterial strains. Furthermore, these procedures can be easily tailored according to the final use of the products, purity degree required, and bacterial strain.

Here, protocols for the isolation of cyanobacterial released carbohydrate polymers and isolation of their exoproteomes are described. Both procedures embody key steps to obtain polymers or proteins with high purity degrees that can be used for further analysis or applications. They can also be easily adapted according to specific user needs.

Cyanobacteria can actively secrete a wide range of biomolecules into the extracellular environment, such as heteropolysaccharides and proteins. The ...

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.

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 ...

Isolation of Active Caenorhabditis elegans Nuclear Extract and Reconstitution for In Vitro Transcription

Caenorhabditis elegans has been an important model system for biological research since it was introduced in 1963. However, C. elegans has not been fully utilized in the biochemical study of biological reactions using its nuclear extracts such as in vitro transcription and DNA replication. A significant hurdle for using C. elegans in biochemical studies is disrupting the nematode&#39;s thick outer cuticle without sacrificing the activity of the nuclear extract. While several methods are used to break the cuticle, such as Dounce homogenization or sonication, they often lead to protein instability. There are no established protocols for isolating active nuclear proteins from larva or adult C. elegans for in vitro reactions. Here, the protocol describes in detail the homogenization of larval stage 4 C. elegans using a Balch homogenizer. The Balch homogenizer uses pressure to slowly force the animals through a narrow gap breaking the cuticle in the process. The uniform design and precise machining of the Balch homogenizer allow for consistent grinding of animals between experiments. Fractionating the homogenate obtained from the Balch homogenizer yields functionally active nuclear extract that can be used in an in vitro method for assaying transcription activity of C. elegans.

Isolation of Active Caenorhabditis elegans Nuclear Extract and Reconstitution for In Vitro Transcription

Here, we describe a detailed protocol for isolating active nuclear extract from larval stage 4 C. elegans and visualizing transcription activity in an in vitro system.

Caenorhabditis elegans has been an important model system for biological research since it was introduced in 1963. However, C. elegans has not been fully ...

Immunopeptidomics: Isolation of Mouse and Human MHC Class I- and II-Associated Peptides for Mass Spectrometry Analysis

Immunopeptidomics is an emerging field that fuels and guides the development of vaccines and immunotherapies. More specifically, it refers to the science of investigating the composition of peptides presented by major histocompatibility complex (MHC) class I and class II molecules using mass spectrometry (MS) technology platforms. Among all the steps in an MS-based immunopeptidomics workflow, sample preparation is critically important for capturing high-quality data of therapeutic relevance. Here, step-by-step instructions are described to isolate MHC class I and II-associated peptides by immunoaffinity purification from quality control samples, from mouse (EL4 and A20), and human (JY) cell lines more specifically. The various reagents and specific antibodies are thoroughly described to isolate MHC-associated peptides from these cell lines, including the steps to verify the beads-binding efficiency of the antibody and the elution efficiency of the MHC-peptide complexes from the beads. The protocol can be used to establish and standardize an immunopeptidomics workflow, as well as to benchmark new protocols. Moreover, the protocol represents a great starting point for any non-experts in addition to foster the intra- and inter-laboratory reproducibility of the sample preparation procedure in immunopeptidomics.

Here, we present a protocol for the purification of MHC class I and class II peptide complexes from mouse and human cell lines providing high-quality immunopeptidomics data. The protocol focuses on sample preparation using commercially available antibodies.

Immunopeptidomics is an emerging field that fuels and guides the development of vaccines and immunotherapies. More specifically, it refers to the science ...

Paper-Based Preconcentration and Isolation of Microvesicles and Exosomes

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Looking Outwards: Isolation of Cyanobacterial Released Carbohydrate Polymers and Proteins

Isolation and Analysis of Plasma Lipoproteins by Ultracentrifugation

Isolation of Active Caenorhabditis elegans Nuclear Extract and Reconstitution for In Vitro Transcription

Immunopeptidomics: Isolation of Mouse and Human MHC Class I- and II-Associated Peptides for Mass Spectrometry Analysis