Urine exosome extraction: Are there any reagents that can handle large-volume samples?

If considering precipitation, use a precipitation solution specifically designed for large-volume urine, rather than directly applying a precipitation reagent intended for cell supernatant. For 50–80 mL, typically incubate overnight at 4°C. After centrifugation, the pellet may appear gelatinous; first resuspend it in a small volume of PBS or the manufacturer's dilution buffer, then perform SEC or wash once with protein wash buffer to reduce contaminating proteins. Dirty EM backgrounds mostly come from uromodulin and salts, which are difficult to avoid with precipitation alone. Before subsequent mass spectrometry, it is recommended to run SDS-PAGE to check for high-abundance bands, and if necessary, add one SEC step.

For 50-80 ml of rat urine, I recommend first performing low-speed centrifugation to remove cells and debris, then concentrating to 5-10 ml using a 100 kDa ultrafiltration tube. Choose a low-adsorption regenerated cellulose membrane for the ultrafiltration tube to avoid excessive adhesion of exosomes; after concentration, extract using SEC or a membrane affinity column, which gives much cleaner background than direct precipitation. For the per-sample cost, if the ultrafiltration tube is amortized over repeated uses, plus the column consumables, it can usually be negotiated to within 100 yuan. Urine is dilute, so add protease inhibitors at collection and process as soon as possible at 4°C to avoid Tamm-Horsfall protein aggregation.

For large-volume, low-abundance samples, batch-to-batch consistency matters more than low cost. When contacting domestic manufacturers, state directly that you are seeking a long-term partnership, request particle size and protein profile data from three trial batches, and confirm that the large-volume urine protocol is in stock, supports tiered pricing, and has stable logistics. Mass spectrometry is sensitive to detergents and salts, so be sure to ask whether the wash buffer is mass spectrometry-compatible. You can first purchase a trial size and use the same batch of rat urine to compare it with your current precipitation method, focusing on the exosomal marker proteins identified by mass spectrometry and the extent to which contaminating proteins are reduced.

We directly use ultracentrifugation, but the volume is too large and it is too tiring. We are also looking for precipitation-based reagents that can handle 100 ml.

Try tangential flow filtration, which can handle large volumes, but you'll need to optimize the recovery rate yourself.