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.
Peak EV isolation
Apex SEC Columns
High performance isolation
- Reproducible performance < 5% CV
- Available with two gel types: 4B (high-purity) & 6B (high-yield)
- Sample volume 0.5-1.0 mL
Negative staining of EVs isolated using Apex 4B column from plasma
How do I apply Apex columns to my research?
Apex 6B Column: Optimized for the highest EV recovery. Ideal for targeted downstream analysis including immunoassays, label-based imaging assays, label-based flow assays, western blots, and pull-down using a specific surface marker.
Apex 4B Column: Optimized for the highest purity. Ideal for unbiased downstream analysis, including proteomics and RNA-seq.
For the best reproducibility and throughput, Apex columns combined with the Ascent instrument can elevate your research.
Apex performance with plasma
- Apex 4B: Optimized for high purity and lowest protein co-elution (ideal for biofluids)
- Apex 6B: Optimized for highest EV yield (ideal for cell cultures)
| Column Type | Purity | Yield |
| Apex 4B | ++++ | +++ |
| Apex 6B | +++ | ++++ |
Apex maintains performance with reuse
Apex maintains performance and drip speed after 5 reuses. Columns were tested by running 0.5 mL of human plasma sample, and pooling fractions 1–3, which represent the EV fractions.
Columns were washed with 4 mL of NaOH, followed by 20 mL of 1X PBS between reuses. EVs and HSA were quantified using the Atlas ELISA
Technical information
The Apex size exclusion column (SEC) purifies extracellular vesicles (EVs) from biological fluids such as plasma, serum, urine, cell culture media, or cerebrospinal fluid (CSF).
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% |
Frequently Asked Questions
How many times can Apex columns be used?
We have tested columns for up to 5 uses.
What is the repeatability of isolation after regeneration cycles?
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.
Should I use Apex 6B or Apex 4B?
Apex 6B (SEC columns packed with Sepharose CL-6B resin) 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 (SEC columns packed with Sepharose CL-4B resin) are optimized for the highest purity. These are Ideal for unbiased downstream analysis, including proteomics and RNA-seq.
References
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.
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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.
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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.
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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.
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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.
