Name: protein digestion, ultrafiltration, and size exclusion chromatography to optimize the isolation of exosomes from human blood plasma and serum
Uploaded: 2018-04-13T15:00:00
Description: Watch this Scientific Journal Video about Protein Digestion, Ultrafiltration, and Size Exclusion Chromatography to Optimize the Isolation of Exosomes from Human Blood Plasma and Serum at JoVE.com

This research was supported by internal funds from CSU (to KMD), ATCC contract #2016-0550-0002 (a subcontract of NIAID HHSN272201600013C), and The Bill and Melinda Gates Foundation (OPP1039688) (to KMD/NKG). We thank the NSF Research Experience for Undergraduates (REU) summer program at Colorado State University for additional support.

This work was determined not to be human subject research upon initial review from Colorado State University&#39;s Institutional Review Board (IRB), as all human samples were obtained as de-identified samples from the Bioreclamation IVT biorepository and were collected under IRB approved protocols.
1. Preparation of Crude Sample (either Plasma or Serum) by Pelleting Larger Vesicles and Cellular Debris
Note: Safety consideration: plasma, serum, and oth...

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An optimized method to increase the purity and yield of exosomes from blood will increase the ability to accurately mine extracellular vesicles as a source of biomarkers for several diseases. The standard methods currently used to isolate exosomes, specifically, ultracentrifugation and precipitation methods, have several disadvantages including exosome aggregation and pelleting of soluble proteins7,17,18. Alternatively, the use ...

Exosomes are nanovesicles (ranging in size from 30 nm to 150 nm) released by almost all the cells in the human body to facilitate cell-to-cell communication processes1,2. Interestingly, the composition of exosomes changes depending on the cells of origin as well as the health status of the individual3,4,5. Additionally, exosomes can be retrieved from several biological fluids such as: saliva, urine, and blood2. Because of these features, exosomes are considered to be a good source of disease biomarkers. Unfortunately, there is no standard method for the isolation of exosomes. Some laboratories consider multistep centrifugation, with a final high-speed step at 100,000 x g using density gradients as the gold standard method for exosome isolation. However, recent studies have shown that ultracentrifugation induces aggregation of exosomes with soluble proteins and other exosomes, in addition to affecting exosome integrity, both of which may hamper downstream applications6,7. Other common methods of exosome isolation include, but are not limited to: precipitation by commercial polyethylene glycol (PEG) based reagents, centrifugal ultrafiltration, and size-exclusion chromatography (SEC). The commercial reagents using polyethylene glycol (PEG) polymers enrich exosomes by causing them to precipitate and form a pellet. Limitations using this polymer are contamination with residual PEG polymer and an abundance of soluble non-exosomal proteins in the final product. Ultrafiltration utilizes centrifugation to purify and concentrate vesicles using a cellulose membrane; exosomes are retained above the filter, while smaller impurities and other proteins pass through the membrane7,8. Just like other methods, centrifugal ultrafiltration has a limited capacity to purify exosomes due to the retention of high levels of non-exosomal proteins, including protein complexes and aggregates. Finally, SEC purification uses porous resin to separate molecules by size. SEC has shown promising results, overcoming most of the problems experienced with other methods by capturing the majority of contaminant proteins and preserving exosomal integrity, since isolation is based on gravity or low-pressure systems7,9. However, the co-isolation of larger protein aggregates and lipoproteins10 during SEC affects the purity of the final exosome preparation. While some methods have been tested for exosome purification from cell culture supernatants and plasma7, or only plasma9,11, there is no information about the performance of methods directly comparing blood plasma and serum from the same individual.
Here, we focus on the purification of blood exosomes by comparing a variety of workflows to determine if vesicle enrichment techniques are translatable between plasma and serum. Nanoparticle tracking analysis and western blot were used to quantify the differences in exosome concentration, purity, and protein composition in the end products. The final method, detailed in this protocol, increases vesicle numbers and reduces non-exosomal protein levels. Importantly, a drastic reduction of common co-precipitating proteins including albumin and apolipoproteins is demonstrated. The high abundance of these two proteins in blood and the frequency at which they co-purify with exosomes causes inconsistencies between &#34;purified&#34; samples, skewing the downstream analyses. This protocol includes the use of a commercially available SEC resin; the resin is composed of porous beads in which proteins smaller than 700 kDa can enter the beads. Once inside, the proteins are retained by an octylamine ligand. The eluate is composed of exosomes and molecules larger than 700 kDa12. Additionally, in order to reduce the protein aggregates and apolipoproteins which evade bead trapping, we include a protease digestion step in the workflow. Currently, there is no single technique capable of maximizing exosome yield while reducing co-purifying non-exosomal proteins. This study shows that a purification protocol that combines a protease digestion pretreatment with multiple recovery and purification methods can be used to increase exosome yield and purity from blood serum and plasma.

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			<td height="21" style="height:21px;width:281px;">Nanosight</td>
			<td style="width:149px;">Malvern</td>
			<td style="width:123px;">NS300</td>
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			<td height="21" style="height:21px;width:281px;">Benchtop microcentrifuge&#160;</td>
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			<td style="width:123px;">Legend Mico21</td>
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			<td height="21" style="height:21px;width:281px;">Benchtop centrifuge&#160;</td>
			<td style="width:149px;">Beckman Coulter</td>
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			<td height="21" style="height:21px;width:281px;">Waterbath&#160;</td>
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			<td height="21" style="height:21px;width:281px;">Microplate reader</td>
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			<td height="21" style="height:21px;width:281px;">Pierce BCA Protein Assay Kit</td>
			<td style="width:149px;">THERMO</td>
			<td style="width:123px;">23227</td>
			<td style="width:124px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">Micro BCA Protein Assay Kit</td>
			<td style="width:149px;">THERMO</td>
			<td style="width:123px;">23235</td>
			<td style="width:124px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">Phosphate buffered saline</td>
			<td style="width:149px;">Corning</td>
			<td style="width:123px;">21-040-CV</td>
			<td style="width:124px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">96 well plate</td>
			<td style="width:149px;">Corning</td>
			<td style="width:123px;">15705-066</td>
			<td style="width:124px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:281px;">Amicon Ultra-0.5 Centrifugal Filter Unit with Ultracel-100 membrane</td>
			<td style="width:149px;">EMD-Millipore</td>
			<td style="width:123px;">UFC510096</td>
			<td style="width:124px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">Amicon Ultra-4 Centrifugal Filter Units</td>
			<td style="width:149px;">EMD-Millipore</td>
			<td style="width:123px;">UFC800324</td>
			<td style="width:124px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">Capto Core 700</td>
			<td style="width:149px;">GE Healthcare</td>
			<td style="width:123px;">17548103</td>
			<td style="width:124px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">Poly-Prep&#160;Chromatography Columns</td>
			<td style="width:149px;">BIORAD</td>
			<td style="width:123px;">7311550</td>
			<td style="width:124px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">NuPAGE 4-12% Bis-Tris Protein Gels</td>
			<td style="width:149px;">THERMO</td>
			<td style="width:123px;">NP0323BOX</td>
			<td style="width:124px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">Nitrocellulose Membrane, Roll, 0.2 &#181;m</td>
			<td style="width:149px;">BIORAD</td>
			<td style="width:123px;">1620112</td>
			<td style="width:124px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">Proteinase K,&#160;Tritirachium album</td>
			<td style="width:149px;">EMD-Millipore</td>
			<td style="width:123px;">539480</td>
			<td style="width:124px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">SimplyBlue SafeStain</td>
			<td style="width:149px;">THERMO</td>
			<td style="width:123px;">LC6065</td>
			<td style="width:124px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">SIGMAFAST&#160;BCIP/NBT</td>
			<td style="width:149px;">Millipore-SIGMA</td>
			<td style="width:123px;">B5655</td>
			<td style="width:124px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">4-Chloro-1-naphthol</td>
			<td style="width:149px;">Millipore-SIGMA</td>
			<td style="width:123px;">C6788</td>
			<td style="width:124px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:281px;">Anti-CD63 Antibody (rabbit anti-human) with goat anti-rabbit HRP secondary antibody</td>
			<td style="width:149px;">SYSTEM BIOSCIENCES</td>
			<td style="width:123px;">EXOAB-CD63A-1</td>
			<td style="width:124px;">Primary and secondary antibodies</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:281px;">ALB Antibody (F-10)</td>
			<td style="width:149px;">SANTA CRUZ BIOTECHNOLOGY, INC.</td>
			<td style="width:123px;">sc-271605</td>
			<td style="width:124px;">Primary antibody</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:281px;">apoB Antibody (C1.4)</td>
			<td style="width:149px;">SANTA CRUZ BIOTECHNOLOGY, INC.</td>
			<td style="width:123px;">sc-13538</td>
			<td style="width:124px;">Primary antibody</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:42px;width:281px;">apoA-I Antibody (B-10)</td>
			<td style="width:149px;">SANTA CRUZ BIOTECHNOLOGY, INC.</td>
			<td style="width:123px;">sc-376818</td>
			<td style="width:124px;">Primary antibody</td>
		</tr>
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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.

The data presented below were obtained using paired plasma and serum samples from five healthy blood donors. To determine the effects of each processing step, five variations of the presented protocol were performed, and exosome recovery and non-exosome protein removal were compared. The methods trialed included: 1) SEC 700 kDa + Ultrafiltration 3 kDa; 2) Ultrafiltration 100 kDa; 3) Proteinase K + Ultrafiltration 100 kDa; 4) SEC 700 kDa + Ultrafiltration 100 kDa, and 5) Proteinase K + Ult...

Watch this Scientific Journal Video about Protein Digestion, Ultrafiltration, and Size Exclusion Chromatography to Optimize the Isolation of Exosomes from Human Blood Plasma and Serum at JoVE.com

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

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 ...

The overall goal of this procedure is to purify vesicles from blood while reducing co-purifying proteins and complexes which can interfere with downstream analysis including nanoparticle tracking and proteomics. This method can help answer key questions such as what are the proteomic changes in serum or plasma vesicles during the disease state as compared to a healthy state. The main advantage of this technique is the increased accuracy in vesicle enumeration.In removing abundant non-vesicle contaminants, we increase the identification of low abundance proteins and the potential biomarkers of disease. The implication of this method extend to the discovery of novel diagnostic biomarkers. It enables more accurate determination of the proteomic composition of extracellular vesicles in complex matrices, like blood.Generally, individuals new to vesicle purification will struggle because there are many published techniques. The reproducability of downstream proteomic analysis is affected by the co-purification of abundant serum proteins. Equilibrate a serum or plasma sample by placing the tube under non-shaking conditions in a four degree Celsius refrigerator overnight.The next day, aliquot 250 microliters of the sample into a microcentrifuge tube using a 1, 000 microliter pipette. Centrifuge the sample at 18, 000 times G for 30 minutes at four degrees Celsius. Using a 200 microliter pipette, remove 200 microliters of the cleared supernatant and add it to a new microcentrifuge tube.Avoid any pelleted material while transferring the supernatant. Quantify the protein content of the sample by BCA assay using the manufacturer's protocol. To perform the assay, dilute the sample one to 50 in one X phosphate buffered saline.Test each sample in duplicate. Prepare a proteinase K solution at a concentration of 50 micrograms per milliliter in PBS by first adding the corresponding amount of proteinase K to a conical tube. Then add the buffer and vortex for 30 seconds three times at room temperature.Next, add 50 microliters of proteinase K solution to the 10 milligram sample aliquot and mix gently by pipetting up and down using a 200 microliter pipette. Incubate the sample at 37 degrees Celsius in a water bath for 30 minutes, placing the tube in a float tube rack. To prevent potential leakage, avoid complete immersion of the tube in the water bath.Following incubation, transfer the sample and rack to a 60 degree Celsius water bath for 10 minutes to inactivate the proteinase K.Next, rinse an ultrafiltration device containing a 100 kilodalton molecular weight cutoff filter with PBS prior to use. Use a 1, 000 microliter pipette to add 500 microliters of PBS to the filter. After spinning the device at 3, 700 times G for five minutes, discard any remaining retentate as well as the eluate.Increase the volume of the sample to 500 microliters by adding PBS. Then pipette the sample into the pre-rinsed ultrafiltration device. Centrifuge the ultrafiltration device in a fixed angle benchtop centrifuge at 3, 700 times G and four degrees Celsius until the sample has reduced to a volume of 50 microliters.Next, add 500 microliters of PBS directly onto the filter membrane. And pipette up and down five times before centrifuging again as before. Transfer the final 50 microliter retentate to a new microcentrifuge tube.Rinse the filter membrane with 200 microliters of PBS, pipetting up and down 10 times. And transfer the wash to the tube. Then, place the filter holder in the inverse position in a new tube.Run a reverse spin recovery for five minutes at 2, 000 times G to retrieve the remaining sample from the membrane. Place the columns in a size-appropriate rack. Add 850 microliters of SEC slurry with beads having a molecular weight cutoff of 700 kilodaltons using a 1, 000 microliter pipette.Uncap the bottom of the column and let the liquid drain into a waste container for five minutes. Once drained, add 10 microliters of PBS to wash the resin. Allow 20 minutes for the wash to drain from the column.Place the concentrated proteinase K treated sample into a 15 milliliter conical tube. And increase the sample volume to five milliliters by adding PBS with a serological pipette. Mix the tube gently by rocking for one minute.Then, slowly apply the sample to the prepared column with a serological pipette. Collect the flow-through in a new 15 milliliter conical tube. Pipette the collected material over the resin again to remove any remaining material below 700 kilodaltons, collecting the flow-through in a new 15 milliliter conical tube.Wash the resin twice by adding one milliliter of PBS each time. Collect the flow-through in a tube. The total final volume will be roughly seven milliliters.Rinse an ultrafiltration device with a three kilodalton molecular weight filter as before. Centrifuge in a swinging bucket rotor at 3, 700 times G and four degrees Celsius. Buffer will pass through the filter and can be discarded.Concentrated exosomes will be retained above the filter. Centrifuge the sample until the volume above the filter is reduced to 200 microliters. Quantify the protein content of the sample by BCA assay using the manufacturer's protocol.Add five micrograms of purified exosomes to one milliliter of PBS. And vortex for 15 seconds on low medium speed. Place the sample in a one milliliter disposable syringe.If available, set the syringe in an automatic syringe pump set to inject the diluted exosome sample at a rate of 30 microliters per minute. Define the script for analysis to run three technical replicates for a minimum of 30 seconds using a constant flow for every replicate. For the analysis, set the capture threshold at five.Finally, determine the soluble protein reduction as described in the text protocol. Representative results of nanoparticle tracking analysis of purified serum vesicles are shown. The vesicle size is roughly 100 nanometers, as expected.Shown here is a Coomassie-stained protein gel comparing the crude serum, pre and post SEC fractions showing the reduction in albumen and other non-vesicle contaminants. A Western blot displays the retention of CD63, a hallmark exosome protein after proteinase K treatment and purification. The proteinase K step is designed to digest soluble proteins.CD63 is a transmembrane protein and less susceptible to proteinase K at the determined concentration and incubation length. Here, a Western blot shows the depletion of albumen, the most abundant protein in blood and a common co-purifying protein after purification. Once mastered, this technique can be done in six hours.After watching this video, you should have a good understanding of how to use centrifuge ultrafiltration, protease digestion, and size exclusion chromatography to obtain vesicles from biological fluids. It's important to perform protein quantitation by BCA or equivalent technique prior to a chromatography and nanoparticle tracking. Results will vary if protein concentrations exceed those noted in this protocol.Don't forget that working with biological fluids can be extremely hazardous and precautions, such as wearing personal protective equipment including gloves, lab coat, and safety glasses should always be taken while performing this procedure. Following this procedure, other methods like liquid chromatography mass spec can be performed in order to determine the exact proteins contained in the vesicles purified. Though this method can provide insight into the composition of blood vesicles, it can also be applied to other sample types, such as urine, CSF, or cell culture supernatants.

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Research

JoVE Journal

Biochemistry

Online Size-exclusion and Ion-exchange Chromatography on a SAXS Beamline

Biological small angle X-ray scattering (BioSAXS) is a powerful technique in molecular and structural biology used to determine solution structure, particle size and shape, and surface-to-volume ratio of macromolecules. The technique is applicable to a very wide variety of solution conditions spanning a broad range of concentrations, pH values, ionic strengths, temperatures, additives, etc., but the sample is required to be monodisperse. This caveat led to the implementation of liquid chromatography systems on SAXS beamlines. Here, we describe the upstream integration of size-exclusion (SEC) and ion-exchange chromatography (IEC) on a beamline, different methods for optimal background subtraction, and data reduction. As an example, we describe how we use SEC- and IEC-SAXS on a fragment of the essential vaccinia virus protein D5, consisting of a D5N helicase domain. We determine its overall shape and molecular weight, showing the hexameric structure of the protein.

The determination of the solution structure of a protein by small angle X-ray scattering (SAXS) requires monodisperse samples. Here, we present two possibilities to ensure minimal delays between sample preparation and data acquisition: online size-exclusion chromatography (SEC) and online ion-exchange chromatography (IEC).

Biological small angle X-ray scattering (BioSAXS) is a powerful technique in molecular and structural biology used to determine solution structure, ...

Human Peripheral Blood Neutrophil Isolation for Interrogating the Parkinson's Associated LRRK2 Kinase Pathway by Assessing Rab10 Phosphorylation

The leucine rich repeat kinase 2 (LRRK2) is the most frequently mutated gene in hereditary Parkinson&#8217; disease (PD) and all pathogenic LRRK2 mutations result in hyperactivation of its kinase function. Here, we describe an easy and robust assay to quantify LRRK2 kinase pathway activity in human peripheral blood neutrophils by measuring LRRK2-controlled phosphorylation of one of its physiological substrates, Rab10 at threonine 73. The immunoblotting analysis described requires a fully selective and phosphospecific antibody that recognizes the Rab10 Thr73 epitope phosphorylated by LRRK2, such as the MJFF-pRab10 rabbit monoclonal antibody. It uses human peripheral blood neutrophils, because peripheral blood is easily accessible and neutrophils are an abundant and homogenous constituent. Importantly, neutrophils express relatively high levels of both LRRK2 and Rab10. A potential drawback of neutrophils is their high intrinsic serine protease activity, which necessitates the use of very potent protease inhibitors such as the organophosphorus neurotoxin diisopropylfluorophosphate (DIFP) as part of the lysis buffer. Nevertheless, neutrophils are a valuable resource for research into LRRK2 kinase pathway activity in vivo and should be considered for inclusion into PD biorepository collections.

Human Peripheral Blood Neutrophil Isolation for Interrogating the Parkinson&#039;s Associated LRRK2 Kinase Pathway by Assessing Rab10 Phosphorylation

Mutations in the leucine rich repeat kinase 2 gene (LRRK2) cause hereditary Parkinson&#8217;s disease. We have developed an easy and robust method for assessing LRRK2-controlled phosphorylation of Rab10 in human peripheral blood neutrophils. This may help identify individuals with increased LRRK2 kinase pathway activity.

The leucine rich repeat kinase 2 (LRRK2) is the most frequently mutated gene in hereditary Parkinson&#8217; disease (PD) and all pathogenic LRRK2 ...

Ion Exchange Chromatography (IEX) Coupled to Multi-angle Light Scattering (MALS) for Protein Separation and Characterization

Ion-exchange chromatography with multi-angle light scattering (IEX-MALS) is a powerful method for protein separation and characterization. The combination of the high-specificity separation technique IEX with the accurate molar mass analysis achieved by MALS allows the characterization of heterogeneous protein samples, including mixtures of oligomeric forms or protein populations, even with very similar molar masses. Therefore, IEX-MALS provides an additional level of protein characterization and is complementary to the standard size-exclusion chromatography with multi-angle light scattering (SEC-MALS) technique.
Here we describe a protocol for a basic IEX-MALS experiment and demonstrate this method on bovine serum albumin (BSA). IEX separates BSA to its oligomeric forms allowing a molar mass analysis by MALS of each individual form. Optimization of an IEX-MALS experiment is also presented and demonstrated on BSA, achieving excellent separation between BSA monomers and larger oligomers. IEX-MALS is a valuable technique for protein quality assessment since it provides both fine separation and molar mass determination of multiple protein species that exist in a sample.

Ion Exchange Chromatography IEX Coupled to Multi-angle Light Scattering MALS for Protein Separation and Characterization

This protocol describes the use of high-specificity ion-exchange chromatography with multi-angle light scattering for an accurate molar mass determination of proteins, protein complexes, and peptides in a heterogeneous sample. This method is valuable for quality assessment, as well as for the characterization of native oligomers, charge variants, and mixed-protein samples.

Ion-exchange chromatography with multi-angle light scattering (IEX-MALS) is a powerful method for protein separation and characterization. The combination ...

Characterization of Proteins by Size-Exclusion Chromatography Coupled to Multi-Angle Light Scattering (SEC-MALS)

Analytical size-exclusion chromatography (SEC), commonly used for the determination of the molecular weight of proteins and protein-protein complexes in solution, is a relative technique that relies on the elution volume of the analyte to estimate molecular weight. When the protein is not globular or undergoes non-ideal column interactions, the calibration curve based on protein standards is invalid, and the molecular weight determined from elution volume is incorrect. Multi-angle light scattering (MALS) is an absolute technique that determines the molecular weight of an analyte in solution from basic physical equations. The combination of SEC for separation with MALS for analysis constitutes a versatile, reliable means for characterizing solutions of one or more protein species including monomers, native oligomers or aggregates, and heterocomplexes. Since the measurement is performed at each elution volume, SEC-MALS can determine if an eluting peak is homogeneous or heterogeneous and distinguish between a fixed molecular weight distribution versus dynamic equilibrium. Analysis of modified proteins such as glycoproteins or lipoproteins, or conjugates such as detergent-solubilized membrane proteins, is also possible. Hence, SEC-MALS is a critical tool for the protein chemist who must confirm the biophysical properties and solution behavior of molecules produced for biological or biotechnological research. This protocol for SEC-MALS analyzes the molecular weight and size of pure protein monomers and aggregates. The data acquired serve as a foundation for further SEC-MALS analyses including those of complexes, glycoproteins and surfactant-bound membrane proteins.

Characterization of Proteins by Size-Exclusion Chromatography Coupled to Multi-Angle Light Scattering SEC-MALS

This protocol describes the combination of size exclusion chromatography with multi-angle light scattering (SEC-MALS) for absolute characterization of proteins and complexes in solution. SEC-MALS determines the molecular weight and size of pure proteins, native oligomers, heterocomplexes and modified proteins such as glycoproteins.

Analytical size-exclusion chromatography (SEC), commonly used for the determination of the molecular weight of proteins and protein-protein complexes in ...

Translating Ribosome Affinity Purification (TRAP) for RNA Isolation from Endothelial Cells In Vivo

Many studies have been limited to using in vitro cellular assays and whole tissues or isolating of specific cell types from animals for in vitro analysis of transcriptome and gene expression by qPCR and RNA sequencing. Comprehensive transcriptome and gene expression analysis of specific cell types in complex tissues and organs will be critical to understand cellular and molecular mechanisms by which genes are regulated and their association with tissue homeostasis and organ functions. In this article, we demonstrate the methodology for isolation of ribosome-bound RNA directly in vivo in the vascular endothelia of animal lungs as an example. The specific materials and procedures for tissue processing and RNA purification will be described, including the assessment of RNA quality and yield as well as real time qPCR for arteriogenic gene assays. This approach, known as translating ribosome affinity purification (TRAP) technique, can be utilized for characterization of gene expression and transcriptome analysis of certain cell types directly in vivo in any specific type in complex tissues.

Translating Ribosome Affinity Purification TRAP for RNA Isolation from Endothelial Cells In Vivo

We present an approach to purify ribosome-bound mRNA from vascular endothelial cells (ECs) directly in mouse brain, lung and heart tissues via EC-specific genetic tag of enhanced green fluorescence protein (EGFP)in ribosomes in combination with RNA purification.

Many studies have been limited to using in vitro cellular assays and whole tissues or isolating of specific cell types from animals for in vitro analysis ...

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.

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 ...

Analysis and Specification of Starch Granule Size Distributions

Starch from all plant sources are made up of granules in a range of sizes and shapes having different occurrence frequencies, i.e., exhibiting a size and a shape distribution. Starch granule size data determined using several types of particle sizing techniques are often problematic due to poor reproducibility or lack of statistical significance resulting from some insurmountable systematic errors, including sensitivity to granule shapes and limits of granule-sample sizes. We outlined a procedure for reproducible and statistically valid determinations of starch granule size distributions using the electrical sensing zone technique, and for specifying the determined granule lognormal size distributions using an adopted two-parameter multiplicative form with improved accuracy and comparability. It is applicable to all granule sizing analyses of gram-scale starch samples, and, therefore, could facilitate studies on how starch granule sizes are molded by the starch biosynthesis apparatus and mechanisms; and how they impact properties and functionality of starches for food and industrial uses. Representative results are presented from replicate analyses of granule size distributions of sweetpotato starch samples using the outlined procedure. We further discussed several key technical aspects of the procedure, especially, the multiplicative specification of granule lognormal size distributions and some technical means for overcoming frequent aperture blockage by granule aggregates.

Presented here is a procedure for reproducible and statistically valid determinations of starch granule size distributions, and for specifying the determined granule lognormal size distributions using a two-parameter multiplicative form. It is applicable to all granule sizing analyses of gram-scale starch samples for plant and food science research. &#160;

Starch from all plant sources are made up of granules in a range of sizes and shapes having different occurrence frequencies, i.e., exhibiting a size and ...

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 ...

Rapid Assessment of Membrane Protein Quality by Fluorescent Size Exclusion Chromatography

During membrane protein structural elucidation and biophysical characterization, it is common to trial numerous protein constructs containing different tags, truncations, deletions, fusion partner insertions, and stabilizing mutations to find one that is not aggregated after extraction from the membrane. Furthermore, buffer screening to determine the detergent, additive, ligand, or polymer that provides the most stabilizing condition for the membrane protein is an important practice. The early characterization of membrane protein quality by fluorescent size exclusion chromatography provides a powerful tool to assess and rank different constructs or conditions without the requirement for protein purification, and this tool also minimizes the sample requirement. The membrane proteins must be fluorescently tagged, commonly by expressing them with a GFP tag or similar. The protein can be solubilized directly from whole cells and then crudely clarified by centrifugation; subsequently, the protein is passed down a size exclusion column, and a fluorescent trace is collected. Here, a method for running FSEC and representative FSEC data on the GPCR targets sphingosine-1-phosphate receptor (S1PR1) and serotonin receptor (5HT2AR) are presented.

The present protocol describes a procedure to perform fluorescent size exclusion chromatography (FSEC) on membrane proteins to assess their quality for downstream functional and structural analysis. Representative FSEC results collected for several G-protein coupled receptors (GPCRs) under detergent-solubilized and detergent-free conditions are presented.

During membrane protein structural elucidation and biophysical characterization, it is common to trial numerous protein constructs containing different ...

Purifying a Fibrinolytic Enzyme from &lt;em&gt;Sipunculus nudus&lt;/em&gt; via Affinity Chromatography

Affinity Purification of a Fibrinolytic Enzyme from Sipunculus nudus

The fibrinolytic enzyme from Sipunculus nudus (sFE) is a novel fibrinolytic agent that can both activate plasminogen into plasmin and degrade fibrin directly, showing great advantages over traditional thrombolytic agents. However, due to the lack of structural information, all the purification programs for sFE are based on multistep chromatography purifications, which are too complicated and costly. Here, an affinity purification protocol of sFE is developed for the first time based on a crystal structure of sFE; it includes preparation of the crude sample and the lysine/arginine-agarose matrix affinity chromatography column, affinity purification, and characterization of the purified sFE. Following this protocol, a batch of sFE can be purified within 1 day. Moreover, the purity and activity of the purified sFE increases to 92% and&#160;19,200 U/mL,&#160;respectively. Thus, this is a simple, inexpensive, and efficient approach for sFE purification. The development of this protocol is of great significance for the further utilization of sFE and other similar agents.

Author Spotlight: Affinity Purification of a Fibrinolytic Enzyme from Sipunculus nudus  

Here, we present an affinity purification method of a fibrinolytic enzyme from Sipunculus nudus that is simple, inexpensive, and efficient.

The fibrinolytic enzyme from Sipunculus nudus (sFE) is a novel fibrinolytic agent that can both activate plasminogen into plasmin and degrade fibrin ...