Size Exclusion Chromatography (SEC) is a commonly used method for extracellular vesicle (EV) and exosome isolation from biological fluids. SEC is easy, reproducible, and provides a high yield of purified EV’s that retain their functionality.
Apex SEC works the same whether you are using a column or plate format.
Apex 4B: Optimized for balanced purity and yield in plasma—well-suited for targeted assays (immunoassays, imaging, Western blot, pull-downs).
Apex 6B: Optimized for maximum EV recovery—best for samples with low-containments like serum-free cell culture media, CSF, and urine.
| Column Type | Purity | Yield | Pore-Size |
| Apex 6B | 1.0 | 1.0 | 20 nm |
| Apex 4B | 5.0 | 0.8 | 35 nm |
| Competitor | 5.0 | 0.3 | 70 nm |
EV composition and size comparison. Super resolution imaging (ONI) and size comparison by NTA and TEM show that Apex 4B and 6B purify EVs with the same tetraspanin composition and size.
Everest Biolabs Atlas EV and human serum albumin (HSA) ELISA kits can be used to optimize EV yield and purity during fraction collection.
| Column volume | 8.75 mL |
| Input sample volume | 0.5 - 1.0 mL |
| Sample types | plasma, serum, urine, CSF, cell culture media |
| Resin types | 4 % or 6 % cross linked agarose beads |
| Exclusion limit | 35 nm or 20 nm |
| Column reproducibility (CV) | 5% |
| Format | Single column or 24-well plate |
We have tested columns for up to 5 uses.
Apex columns have a 5% CV in EV yield for up to 5 uses when measured with the Atlas EV ELISA. We know how important repeatability is, so we developed a tightly controlled process for manufacturing columns to ensure the most repeatable columns.
Apex MM are optimized for maximum EV purity in plasma/serum samples. These are ideal for unbiasesd assays like Mass Spec and flow cytometry. Apex 6B are optimized for the highest total EV recovery. These are ideal for targeted downstream analysis, such as immunoassays, label-based imaging assays, label-based flow assays, western blots, and pull-down using a specific surface marker. Apex 4B are optimized for the highest purity. These are Ideal for unbiased downstream analysis, including proteomics and RNA-seq.
No, the plate seal can be cut to use only 4 wells at one time and the Summit instrument allows skipping used wells so you can maximize the utility of each and every Apex plate.
High-Throughput Extracellular Vesicle Isolation Using Plate-Based Size Exclusion Chromatography and Automation pubs.acs.org/doi/10.1021/jacs.4c17948
Ter-Ovanesyan, Norman, et al. Framework for rapid comparison of extracellular vesicle isolation methods. eLife 2021 doi.org/10.7554/eLife.70725
This study presents a standardized framework for assessing the efficiency and purity of different extracellular vesicle (EV) isolation techniques.
The authors utilized ultrasensitive single-molecule array (Simoa) assays to quantify three key EV transmembrane proteins-CD9, CD63, and CD81- while measuring albumin levels as a marker of free protein contamination. By applying this approach to plasma and cerebrospinal fluid (CSF), they systematically compared commonly used isolation methods, including ultracentrifugation, precipitation, and size exclusion chromatography (SEC). The results highlight SEC as a superior method for maintaining both yield and purity, particularly when optimized with custom column parameters. This study provides a valuable, reproducible strategy for improving EV isolation, aiding biomarker discovery and translational research in EV-based diagnostics.
Ter-Ovanesyan, Gilboa, et al. Improved isolation of extracellular vesicles by removal of both free proteins and lipoproteins. eLife 2023 doi.org/10.7554/eLife.86394
This study presents an advanced method for isolating extracellular vesicles (EVs) from plasma by minimizing contamination from free proteins and lipoproteins, which traditionally complicate EV purification.
The researchers developed a digital ELISA assay targeting ApoB-100, a key lipoprotein marker, and integrated it with existing assays for albumin and EV-associated tetraspanins. They systematically evaluated various size exclusion chromatography (SEC) resins and developed a novel approach, Tri-Mode Chromatography (TMC), to enhance EV purity while maintaining yield. The study highlights the advantages of TMC in reducing co-isolated contaminants and improving the reliability of EV-based biomarker discovery, particularly for proteomics applications.
Gilboa, Ter-Ovanesyan, et al. Measurement of α-synuclein as protein cargo in plasma extracellular vesicles. PNAS 2024 doi.org/10.1073/pnas.2408949121
This study addresses the challenge of measuring α-synuclein, a key protein associated with Parkinson’s disease (PD), within extracellular vesicles (EVs) isolated from plasma.
Given the difficulty of distinguishing EV-associated proteins from free plasma proteins, the researchers developed a method combining optimized size-exclusion chromatography (SEC) for EV isolation with a protease protection assay and ultrasensitive digital ELISA (Simoa) measurements. Their analysis revealed that only a small fraction of total plasma α-synuclein is contained within EVs, but its phosphorylated form (pSer129), a marker of PD pathology, is enriched within EVs compared to free plasma protein. Applying this method to patient samples, they observed subtle but significant differences in EV α-synuclein and pSer129 levels between PD, Lewy body dementia (LBD), and control groups. This work establishes a robust framework for studying EV-contained neurodegenerative biomarkers and highlights the potential of EV-based diagnostics for neurodegenerative diseases.
Ter-Ovanesyan, et al. Identification of markers for the isolation of neuron-specific extracellular vesicles. bioRxiv 2024 doi.org/10.1101/2024.04.03.587267
This study presents a systematic approach for identifying neuron-derived extracellular vesicle (EV) markers, facilitating the selective isolation of neuron-specific EVs from cerebrospinal fluid (CSF) and plasma.
Researchers developed a framework that integrates gene expression data with EV proteomics to identify transmembrane proteins unique to neurons. They optimized high-purity EV isolation by combining multiple purification techniques, including size exclusion chromatography (SEC), density gradient centrifugation (DGC), and Mixed Mode Resin (MMR) Slurry, to effectively remove free proteins and lipoprotein contaminants while preserving EV integrity. Through proteomic analysis, they identified NRXN3 as a robust neuron-specific EV marker and validated its presence using ultrasensitive immunoassays. By optimizing immuno-isolation protocols, the study provides a foundation for isolating neuron-derived EVs, enabling their use in biomarker discovery for neurological diseases. This methodology offers a scalable strategy for isolating cell type-specific EVs, expanding potential applications in liquid biopsy and neurodegenerative disease diagnostics.
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