Sample collection (per Dr. Robert Star)
- Urine collection solution (volume per tube 50 ml)
- 1.67 ml 100 mM NaN3
- 2.5 ml PMSF (2 mg/ml in isopropyl alcohol, stable 4°C for several months)
- 50 µl Leupeptin (1 mg/ml in ddH2O, stable 1 wk 4°C, 6 mo -20°C)
- Isolation solution (50 ml)
- 10 mM Triethanolamine (MW 185.7) 0.093 g
- 250 mM Sucrose (MW 342.3) 4.28 g
- ddH2O to 45 ml
- Adjust pH to 7.6 with 1N NaOH ~ 220 µl
- Add ddH2O to 50 ml
- Add protease inhibitors day of isolation 2.5 ml PMSF, 50 µl Leupeptin
- 5X SDS-Laemmli (Optional: for western blot)
- SDS 3.75 g
- glycerol 15 ml
- 1 M Tris, pH 6.8 2.5 ml
- Bromophenol Blue dab
- dd-H20 to 50 ml
Starting urine volume
; Our experience: 100-200 ml of urine per patient is enough to get an adequate amount of exosomes for western blot of AQP2, NKCC2, NCC, TSG101, and ALIX.
For western blot
- 17,000 x g spin step to remove whole cells, large membrane fragments, and other debris.
In our hands: Spin the urine samples by Sorvall RC2-B refrigerated centrifuge: 30 ml in each SS-34 tube, 12000 rpm (17000 x g), 10 min, 4°C.
*** Optional Step: If the supernatant is still cloudy, collect the supernatant then repeat spin at above setting.
- 200,000 x g spin step to pellet the exosomes.
*** In our hands: Transfer 8 ml of 17000 x g supernatant to each high speed tube (thick wall, polycarbonate tube #355630, Beckman), match weight. Ultracentrifuge the supernatant with Beckman L8-70M ultracentrifuge using 70.1 Ti rotor, at 47,500 rpm (200,000 x g), 1 hour, 4°C, and vacuum.
*** Optional Step: To increase the yield of exosomes, discard supernatant, transfer another 8 ml of 17000 x g supernatant to the same high speed tube, vortex then repeat ultracentrifugation at above setting. Repeat this optional step 1-2 more times.
- Resuspend the exosome pellet.
In our hands: Discard supernatant, resuspend the first tube’s pellet with 50 µl isolation solution, vortex 30 sec then transfer the suspension (as much as possible) to resuspend second tube and so on for every tube.
*** Optional Step to remove Tamm-Horsfall protein (THP) from the exosomes. Discard supernatant, for each tube add 50 µl isolation solution, vortex 30 sec to resuspend the pellet then transfer suspension (as much as possible) and pool to an Eppendorf tube. Add DTT to the sample to a final concentration of 200 mg/ml, then incubate at 95°C for 2 min to denature the zona pellucida (ZP) domains in the THP, thus inhibiting aggregation and allowing THP to be removed in the supernatant. Transfer the suspension to a high speed tube, fill up isolation solution to 8 ml and repeat ultracentrifugation. Discard supernatant, resuspend the pellet in 50 µl isolation solution.
- Prepare 5X SDS-Laemmli with dithiotrietol (DTT) (60 mg/ml).
- Add 5X SDS-Laemmli-DTT to suspension (1:4), vortex thoroughly.
- Heat samples on heat block at 60°C 10 min, then store in -80°C.
Last Updated: 8/4/2005