• Low exosome yield
• Protein degradation
• Inefficient protein extraction
• Interference from the isolation reagent

To troubleshoot:

• Concentrate your sample further before loading
• Ensure your lysis buffer is compatible with SDS-PAGE
• Consider using Western blotting to detect specific exosomal markers (e.g., CD63, CD81, CD9)

Additional suggestions:

• Verify the presence of exosomes using other methods such as nanoparticle tracking analysis (NTA) or transmission electron microscopy (TEM)
• Consider alternative isolation methods, such as size-exclusion chromatography or density gradient ultracentrifugation, which may provide purer exosome preparations.

Thank you, Dr. Jaishree. I also tried to lyse exosomes with 8M urea + 0.1 % SDS, as I read somewhere that an increased percentage of SDS might interfere with downstream analysis like proteomics. I also tried to precipitate lysed exosomal protein (8M+2 % SDS) with chilled acetone overnight, but I did not get any protein after the Bradford assay in either of the two protocols.

Please suggest any other gentle lysis buffer or a way to reduce protein contamination. It would be very helpful as I am preparing my samples for proteomic analysis.

Thanks

1. Firstly, I am not getting any pellets even after following the manufacturer's instructions.

Follow the structure procedure strictly and trust the kit's versatility. These protocols are strictly validated before commercialization. If the samples have enough exosomes to be precipitated and if nothing in your sample hampers its activity, set aside the kit incompatibility. You may also use different isolation methods such as centrifugation, ultrafiltration, SEC, TFF, immunoaffinity pull-down, etc...Confirm the exosome quality and quantity always just before the protein assay, SDS-PAGE, lysis, or characterization. Step-by-step confirmation may lead you to success without time and fund losses. Use, NTA, TEM, Western identification of exosomal membrane proteins, and SEC-MALLS assays for this purpose.

2. I lysed it with 8M urea and 2% SDS and got 30ug/ul protein after estimation with Bradford assay. Is this correct?

Bradford assay is incompatible to use with the mentioned SDS and urea conditions. You are probably getting false positive results. Try to dilute the sample to reduce the urea and SDS concentration. If your SDS results do not show any protein band, as expected your protein Bradford assay is deceiving you. Use reagent blank also for blank subtraction or to identify misapplication.

3. I am not getting any band when I run it on 12% SDS page.

Do you see protein marker ladder? If the ladder appears but not your proteins this indicates either your lysis procedure is inefficient or not enough exosomes you gained before loading the PAGE. Since SDS is the final step abovementioned confirmation will clarify the subject. Using RIPA and protease inhibitor cocktails and mechanical disruption will break open the exosomes efficiently. Protease inhibitors are critical to keep intact your proteins before downstream assays. You do not need to hesitate to use SDS for proteomic downstream assays because you may get rid of it by employing the well-established FASP protocol.

Hiral Thacker I will suggest using a plasma or serum as control for your experiment. First, you precipitate exosomes using the same kit and do protein estimation. If you are getting protein in good amounts, you can run your gel with 10-20 ug total protein and stain to visualise. In this way you can check your procudure, because plasma will definitely give a good pellet of exosomes.