The process of Western blotting involves several steps that need to be carefully executed for optimal results.
The first step is lysis, which requires lysing the extracellular vesicles (EVs) using radioimmunoprecipitation assay buffer (RIPA) or other methods. EVs can be tougher to lyse than cells, so a higher concentration of RIPA can improve lysis, but it may affect the movement of proteins into the gel due to increased salt concentrations. The ideal RIPA concentration can be titrated using non-precious samples. Additionally, heating samples to 95ºC for 10 minutes can improve lysis.
The second step involves determining how much sample to use. One option is to load an equal volume of sample in each well. Alternatively, you can load an equal amount of protein. If the samples are not sufficiently concentrated, the qEV Concentration kit can be used to concentrate samples into a pellet which can then be resuspended in just the right amount of RIPA needed for your Western blot.
The third step is to decide whether or not to reduce the proteins. Some antibodies will only detect non-reduced forms of the protein, whereas others will only detect the reduced form. Generally, cytosolic proteins (e.g. TSG101) may require reducing conditions whereas membrane-spanning proteins (e.g. tetraspanins) may not.
Finally, you can run the gel and transfer it to a membrane – this part of the process requires careful execution for optimal results. Therefore, it’s important to run a positive control (either purified protein or cell lysate) to ensure that your protocol is working. To test that you have protein and that it has migrated in the gel, you can use a reversible gel stain such as Ponceau S. If protein is present and has run down the gel, you should see a ladder-like protein stain in each well.
Interpretation can be the trickiest step. If you are not seeing the same proteins when using Gen 2 columns for isolating EVs as you did with Legacy columns, it could be due to changes in protocol or increased purity resulting from Gen 2 columns removing more non-EV associated proteins. The Gen 2 columns remove a slightly higher percentage of soluble protein, which may result in purer isolates, meaning that what you thought was an EV protein previously could be a soluble protein. For this reason, it is important to check the protein-rich fractions for your protein of interest.
For a more in-depth guide to Western Blot troubleshooting, read our expanded article on the topic.