Recommended western blot primary and secondary antibody antibodies combinations:įinal Primary Antibody Concentration (µg/mL)
If using semi-dry transfer, use 10 V for 30 minutes.Assemble transfer sandwich as per instrument instruction and then transfer the proteins onto a PVDF membrane.Incubate gel in cold Transfer Buffer for 15 minutes. 35 µL) of samples per well in a 10% polyacrylamide gel. Heat the samples at 95☌ for 5 minutes.In this case, it is recommended to run a small volume of the Positive Selection Cocktail to check for background signals. If you used EasySep™ to isolate the EVs, the antibodies from EasySep™ Positive Selection Cocktail may be detected by secondary antibodies.To prepare 4X Laemmli Sample Buffer with reducing agent, add 100 µL of 2-mercaptoethanol to 900 µL of 4X Laemmli Sample Buffer, or add dithiothreitol to 4X Laemmli Sample Buffer to a final concentration of 50 mM. When uncertain, test 4X Laemmli Sample Buffer with and without reducing reagent. EV markers other than CD9/CD63/CD81/CD45/EpCAM may require a reducing agent added to Laemmli Sample Buffer.Note: When blotting for CD9, CD63, CD81, EpCAM, and CD45, 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. 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. Tetraspanin markers such as CD9, CD63, and CD81 are proteins commonly found on EVs across different cell types.
Isolated EVs can be characterized by western blotting (also referred to as immunoblotting), a widely used technique to detect specific protein markers in a sample. Once isolated, the particles should 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. Using EasySep™ Human Extracellular Vesicle Positive Selection Kits or Extracellular Vesicle SEC Columns, researchers can easily isolate and purify human EVs from biofluids-including serum and plasma-and from culture-conditioned media.
UML SEQUENCE DIAGRAM SYMBOLS PLUS
MesenCult™-ACF Plus Medium), such as immunomagnetic separation, differential ultracentrifugation, or size exclusion chromatography (SEC)*. There are several ways for isolating EVs from biofluids or cell culture conditioned media (e.g. Due to their inherently heterogeneous nature as well as the complexity of biological samples, it is recommended to characterize EVs after isolation. These vesicles, carrying protein and genetic cargo, play an important role in intercellular communication and are recognized for their potential therapeutic applications. Tissue and Cell Culture Dissociation ReagentsĮxtracellular vesicles (EVs) are lipid bilayer-enclosed structures released by almost all cell types.Work at STEMCELL View Current Opportunities >
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