How to Characterize Extracellular Vesicles by Western Blotting
Extracellular vesicles (EVs) are lipid bilayer-enclosed, extracellular structures released by almost all cell types. These vesicles, carrying protein and genetic cargo, play an important role in intercellular communication and are recognized for their potential therapeutic applications. Due to their inherently heterogeneous nature as well as the complexity of biological samples, it is recommended to characterize EVs after isolation.
There are several ways for isolating EVs from biofluids or cell culture conditioned media (e.g. MesenCult™-ACF Plus Medium), such as immunomagnetic separation, differential ultracentrifugation, or size exclusion chromatography (SEC)*. Using EasySep™ Human Extracellular Vesicle Positive Selection Kits, researchers can easily isolate and purify human EVs from biofluids—including serum and plasma—and from culture-conditioned media. Once isolated, the isolated particles can be analyzed to confirm that they are indeed EVs and not products of cell fragmentation or other contaminants such as protein complexes or lipoproteins, usually present in biological samples.
Isolated EVs can be characterized by western blotting or immunoblotting, a widely used technique to detect specific protein markers in a sample. Tetraspanin markers such as CD9, CD63, and CD81 are proteins commonly found on EVs across different cell types. Here, we provide a detailed protocol for performing a western blot to detect the presence of such characteristic EV-associated proteins in order to confirm the presence of EVs in the biological sample.
* For information regarding isolation of EVs using EasySep™, refer to the Product Information Sheets (PIS) for EasySep™ Human Pan-Extracellular Vesicle Positive Selection Kit, EasySep™ Human Extracellular Vesicle (CD81) Positive Selection Kit, EasySep™ Human Extracellular Vesicle (CD9) Positive Selection Kit, or EasySep™ Human Extracellular Vesicle (CD63) Positive Selection Kit. For generating EVs from mesenchymal stromal cell (MSC)-conditioned medium, refer to this technical bulletin.
- 4X Laemmli Sample Buffer (Bio-Rad Catalog #1610747)
- Tris-hydrochloride (Tris-HCl)
- Distilled water (dH2O)
- 10% Sodium dodecyl sulfate (SDS) in dH2O
- Methanol (MeOH)
- Bovine serum albumin (BSA)
- TWEEN® 20 See More
- D-PBS (Without Ca++ and Mg++) (Catalog #37350)
- Low-binding microcentrifuge tube (Catalog #38037 or 38090)
- Reducing agent (optional; e.g. 2-mercaptoethanol or dithiothreitol)
- 10% polyacrylamide gel for electrophoresis
- Polyvinylidene difluoride (PVDF) membrane
- Horseradish peroxidase (HRP)- or fluorochrome-conjugated primary antibodies
I. Buffer Preparation
- Prepare Transfer Buffer by combining the components in the order listed in Table 1. Mix thoroughly.
Table 1. Preparation of Transfer Buffer
ComponentFinal ConcentrationAmount to Prepare 1LTris-HCl48 mM5.8 gGlycine39 mM2.9 g10% SDS0.0375%3.75 mLdH2O-800 mLMeOH20%200 mL
- Prepare Blocking Buffer by adding the components listed in Table 2. Mix the solution thoroughly after adding each component.
Table 2. Preparation of Blocking Buffer
ComponentsFinal ConcentrationAmount to Prepare 10 mLBSA5%0.5 gTWEEN® 200.2%20 µLPBS-10 mL
- Prepare Wash Buffer by adding 1 mL of TWEEN® 20 in 1 L PBS.
- Isolate EVs using EasySep™ Human Extracellular Vesicle Positive Selection Kits.
- Fully resuspend EVs in residual liquid from the isolation process and adjust to the desired volume. For instance, EVs isolated from 1 mL of plasma can be adjusted to a final volume of 250 µL. Store isolated EVs in a low-binding tube to reduce EV loss to the sample tube. Set aside 30 µL for western blot analysis.
- Dilute 3 parts sample with 1 part 4X Laemmli Sample Buffer.
Note: When blotting for CD9, CD63, and CD81 antibodies, do not add a reducing agent to the Laemmli Sample Buffer, as CD9/CD63/CD81 detection antibodies often recognize the disulfide bond on the antigen's epitope.
- Heat the samples at 95°C for 5 minutes.
- Load the desired volume (e.g. 35 µL) of samples per well in a 10% polyacrylamide gel.
- Run SDS-PAGE for 30 minutes at 200 V.
- Rinse gel twice with distilled water. Incubate gel in cold Transfer Buffer for 15 minutes.
- Assemble transfer sandwich as per instrument instruction and then transfer the proteins onto a PVDF membrane.
Note: Use a low-fluorescence PVDF membrane or nitrocellulose membrane for fluorescence imaging.
- Rinse the membrane twice with PBS and incubate with Blocking Buffer at room temperature for 1 hour.
- Wash the membrane 2X for 3 - 5 minutes each with Wash Buffer.
- Dilute primary antibodies in the blocking buffer.
Recommended dilution ranges:
- Incubate the membrane with HRP- or fluorochrome-conjugated primary antibodies at room temperature for 1 hour.
- Wash the membrane 3X for 3 - 5 minutes each with Wash Buffer.
- To detect signals from HRP-conjugated antibodies, incubate blot with enhanced chemiluminescence (ECL) substrate and image. To detect a signal from fluorochrome-conjugated antibodies, use fluorescence imaging.
II. EV Sample Preparation
IV. Immunoblotting and Imaging
Figure 1. Typical Western Blot Analyses of EVs Isolated from Human Plasma and Mesenchymal Stromal Cell (MSC)-Conditioned Medium
The western blot analyses in the above examples show positive isolation of EVs with CD9, CD63, and CD81 tetraspanin markers from (A) human plasma and (B) MSC-conditioned medium using EasySep™ Human Pan-Extracellular Vesicle Positive Selection Kit (Catalog # 17891). Western blots were run under non-reducing conditions.
Highly pure human EVs can be easily obtained—and in as little as 30 minutes—with EasySep™ using immunomagnetic separation. Explore all our products for EV isolation and characterization below.
Products for Isolation of EVs Using Immunomagnetic Separation
Products for Characterization of EVs
- Kowel EJK et al.(2017) Extracellular vesicle isolation and analysis by western blotting. Methods Mol Biol. 1660:143–52.