Vesicle Kit Instructions

Urine Extracellular Vesicle Isolation Kit Instructions

Component

Contents

Storage

Treatment Buffer

10 mL

4C

Conditioning Buffer

4 mL

Room Temp

Precipitation Buffer

4 x 1 mL

-20C

Elution Buffer

2 x 5 mL

Room Temp

Storage Conditions and Product Stability

All solutions should be kept tightly sealed and stored at the indicated temperature. If any precipitation occurs in the Precipitation Buffer, heat to 95C for 5 minutes and invert. Repeat until the precipitate is mostly dissolved. Small residual precipitation will not affect performance. These reagents should remain stable for at least 1 year in their unopened containers.

Precautions and Disclaimers

This kit is designed for research purposes only. It is not intended for human or diagnostic use. Ensure that a suitable lab coat, disposable gloves, and protective goggles are worn when working with chemicals. If liquid containing these buffers is spilled, clean with suitable laboratory detergent and water. If the spilled liquid contains potentially infectious agents, clean the affected area first with laboratory detergent and water, and then with 1% (v/v) sodium hypoclorite.

Protocol (sample volume of <10mL):

Note: Ensure that all solutions are at room temperature prior to use. If precipitates have formed, warm the solutions and mix well until the solutions become clear again.

  1. Add 0.1 volume of Treatment Buffer to Whole Urine
    Example: for 2mL of urine add 0.2ml of Treatment Buffer; for 4ml of urine add 0.4ml Treatment Buffer
  2. Centrifuge at 1,500xg for 5min at RT. Transfer the supernatant to a new tube.
  3. Add 0.04 volume of Precipitation Reagent. Mix by inverting the tube 3 times.
    Example: for 1mL of urine add 0.04ml of Precipitation Reagent; for 3ml of urine add 0.12ml Precipitation Reagent
  4. Add 0.04 volume of Conditioning Buffer. Mix by inverting the tube 3 times.
    Example: for 1mL of urine add 0.04ml of Conditioning Buffer; for 3ml of urine add 0.12ml Conditioning Buffer
  5. Incubate on ice for 10min.
  6. Centrifuge at 3,000xg for 3min at RT. Discard the supernatant. Resuspend the pellet in a volume of Elution Buffer in a manner consistent with your downstream analysis (see below)

For RNA

  • Note: Elution Buffer is compatible with most RNA Purification Kits. However, for best results, we recommend the Ymir Urine Extracellular RNA Purification Kit.
  • Add 0.35mL of Elution Buffer to the precipitate and pipette up and down until completely dissolved.

    If the pellet does not completely dissolve increase the amount of Elution Buffer until the entire pellet is back in solution.

For Immunoblot Analysis

  • Add 0.1 volume of the starting volume of Elution Buffer to the precipitate and pipette up and down until partially or completely dissolved. Heat at 95C for 5min. Centrifuge briefly to remove any precipitate.

    Example: for 1mL of urine add 0.1ml of Elution Buffer; for 3ml of urine add 0.3ml Elution Buffer. Rarely, the pellet will not completely dissolve; a small amount of residual pellet can be spun away without significant loss in yield, however, some users prefer to increase the amount of Elution Buffer until the entire pellet is back in solution.

For Intact Extracellular Vesicles (Microscopy, Nanosight, Immunoblot Analysis)

  • Add 0.1 volume of the starting urine volume of Elution Buffer to the precipitate and pipette up and down until completely dissolved.

    Example: for 1mL of urine add 0.1ml of Elution Buffer; for 3ml of urine add 0.3ml Elution Buffer. Rarely, the pellet will not completely dissolve; a small amount of residual pellet can be spun away without significant loss in yield, however, some users prefer to increase the amount of Elution Buffer until the entire pellet is back in solution.

A note on storage of eluted vesicle preps:

Eluted vesicles in 1x Elution Buffer are stable for at least up to a week at 4C and for at least a month at -80C. We don’t recommend storage at -20C for more than a week. If a precipitate appears during storage simply heat to 37C and gently pipette up and down a few times to resuspend.