Extracellular Vesicle (Ev) SEC Columns 70 nm

Product details

It is recommended to use buffers with an ionic strength of 0.15 M or higher, because in low ionic strength buffers, the concentration of competing buffer ions is low, and contaminant proteins spend most of their time adsorbed to binding sites on the stationary phase. For buffer choice it is also important consider the downstream protocol for isolated vesicles. For example citrate buffers could interfere with molecular biology protocols.

Note: All buffers must contain sodium azide.

Columns are reusable up to 5 times and should not be used if any evidence of deterioration is observed.

PROTOCOL
1. Place the column in a rack and level it, make sure the column is vertical.
2. Remove the top cap carefully, as drastic changes in pressure could damage the column.
3. Remove the bottom cap.

Column Equilibration
4. The column must be at room temperature (18ºC – 24ºC) for 30 minutes before use.
5. Use only fresh filtered (0.2 μm) buffer to avoid particulate contamination.
6. Rinse the column with 21 mL of running buffer (7,5 mL for the lower volumes columns).
7. Do not allow the column to run dry. The top filter must stay wet.

Sample fraction collection
8. Once the column is washed, add the sample onto the top filter (500 μL, and 150 μL for the lower volumes columns). Once it passes through the filter, fill the column with the running buffer.
9. Let pass three drops of running buffer, and collect fractions of 500 μL (150 μL for the lower volumes columns) (minimum 12 fractions).
10. When the column is used according to the protocol, the first four fractions (2.0 mL, and 60 0 μL for the lower volumes columns) is the void volume which does not contain vesicles. These vesicles elute predominantly in fractions 5, 6, 7 and 8 with removal of protein contamination. Fractions beyond 9 usually contain higher protein and lower vesicle levels.

COLUMN CLEANING AND STORAGE
1. After the collection of the vesicle fraction, rinse the column with 21 mL (7,5 mL for the lower volumes columns) of running buffer to rinse out all the protein and small molecules before the next sample application.
2. NaOH cleanup is performed to remove precipitated proteins or other contaminants that may be accumulated on the column, a running buffer change, or a sample change. To do this, add 0.5mL (0,15 mL for the lower volumes columns) of sodium hydroxide (500 mM NaOH), then equilibrate the columns with 10 mL of water (3,5 mL for the lower volumes columns) followed by 21 mL of running buffer (7,5 mL for the lower volumes columns) immediately after cleanup.
Note that the column should never be stored in sodium hydroxide.
3. Put the bottom cap on.
4. Put the top cap on carefully, as drastic changes in pressure could damage the column.
5. Stopcocks cannot be heat sterilized as they do not withstand high temperatures. To sterilize them, it can be done by passing a 30% ethanol solution (5 mL) with the help of a syringe.

FRACTION COLLECTION CONCENTRATION
If the volume of the starting sample is small and / or the concentration and EVs is low in the sample, it is possible that the EVs will be much diluted in the collected fractions. In many cases, for downstream processing it is necessary to concentrate the fractions.