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Extracellular vesicle research bottleneck? Simple Western doubles detection efficiency
Date: 2025-10-21Read: 0
ExosomesIt is a small lipid nanoparticle derived from endosomes, with a diameter of about 30-200 nanometers. Most living cells actively secrete exosomes through exocytosis[1]Whether in pathological or physiological states, the release of extracellular vesicles can be persistent or inducible, and the release process is dynamic, controllable, and functionally correlated. The molecular composition and release amount of extracellular vesicles depend on the state of their source cells. Extracellular vesicles can be isolated from various cell lines and primary cultures, as well as extracted from biological fluids such as serum, plasma, and saliva. In view of the diversity of sources, exosomes have multiple physiological functions, and more and more studies show that they play an important role in many pathological conditions, such as cancer, infectious diseases, neurodegenerative diseases, etc. Therefore, extracellular vesicles have become a key research object in liquid biopsy methods[2]In addition, extracellular vesicles can also be engineered to possessPotential drug delivery vehicles[3].
Due to the small volume of extracellular vesicles, isolating them from complex biological matrices is quite challenging, and the isolation yield is usually low.In this context, Simple Western ™ Becoming an ideal technique for studying extracellular vesicles: This method requires only 3 microliters of sample and has Pick level sensitivity, significantly reducing the requirements of traditional Western blot for the required amount of extracellular vesicles.Subsequently, specific detection antibodies targeting extracellular vesicle associated antigens can be used for characterization analysis - these target antigens can be either universal markers of extracellular vesicles or all markers of their source cells or body fluids. In this experimental protocol, we will use commercially available extracellular vesicle standards and antibodies to perform Simple Western blotting ™ Universal and source specific biomarkers for detecting extracellular vesicles.
This article introduces the results of verifying the effectiveness of the antibodies in the table using three extracellular vesicle standards: human plasma, human serum, and human A549 cells (alveolar basal epithelial adenocarcinoma cells)
01. Expression of universal extracellular vesicle markers
Flotilin-1: As a classic universal biomarker, it was detected in all three standard samples (Figure 1A).
Alix is expressed in all three standard samples, but the expression level is higher in A549 cell-derived exosomes (Figure 1D).
02. Expression of source specific biomarkers
Annexin V: As an apoptosis marker and blood anticoagulant protein, it was detected in A549 cells and plasma derived exosomes, but not in serum derived exosomes (Figure 1B).
EGFR (HER1): Only detected in A549 cell-derived exosomes, with no signal observed in serum and plasma derived exosomes (Figure 1C).
Figure 1: Using Simple Western ™ Measure the target antigen of extracellular vesicles. A：flotillin-1； B：Annexin V； C：EGFR； D：Alix。
03. Signs of special processing requirements
CD63: The signal can only be detected when the exosome standard is treated with PNGase F (deglycosylation enzyme), indicating the influence of glycosylation modification on the detection of this marker (Figure 2).
04. Verification of other biomarkers
The remaining antibodies in Table 2 (such as Annexin A2, HSPA8/HSC70, TSG101, etc.) were validated by Simple Western and the target signals were detected in the corresponding exosome standards (Figure 2).
Figure 2. View of detection lanes for each target antigen in plasma and A549 cell-derived extracellular vesicle standards.
——The core value of Simple Western technology——
(1) Micro high efficiency:Only 3-5 μ L of extracellular vesicle lysate is needed (traditional WB requires 50 μ L+), suitable for precious samples; The entire capillary process reduces sample loss and is suitable for low abundance membrane proteins.
(2) High sensitivity: Pick level detection limit, capable of detecting CD63/Alix, etc. After PNGase F treatment, glycosylation interference is resolved to achieve specific detection of EGFR and other substances.
(3) Anti matrix interference: 12-230kDa capillary electrophoresis for impurity removal. After extracellular vesicle lysis, it can be directly loaded onto the machine without complicated purification.
(4) Multiple quantification: Single sample synchronous measurement of 8 targets, meeting MISEV standards, with a dynamic range covering 6 orders of magnitude.
(5) Automated process: Fully automated results within 3 hours to reduce protein degradation; Each well has independent quality control to meet the QC requirements for extracellular vesicle preparation.
(6) Accurate quantification: supports absolute/relative quantification, low sample size+high repeatability, adapts to the development of liquid biopsy markers, accelerates the translation of scientific research into clinical practice.
*This article is reproduced from "ProteinSimple" WeChat official account
*Reference:
1. Exosomes, D M Pegtel and S J Gould, Annual Review of Biochemistry, 2019, 88:487–514.
2. Extracellular vesicles – biomarkers and effectors of the cellular interactome in cancer, J Rak, Frontiers in Pharmacology, 2013, 4:21.
3. Exosomes as reconfigurable therapeutic systems, R Conlan, S Pisano, M Oliveira, M Ferrari and I Mendes Pinto, Trends in Molecular Medicine, 2017, 23(7):636–650.
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