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A Size Exclusion Chromatography Technique to Purify Mycobacterial Extracellular Vesicles


Allow the size exclusion chromatography, or SEC column, to equilibrate to room temperature. Turn on the AFC using the power switch on the back of the tower unit, and press SETUP on the main menu touchscreen if necessary to adjust the settings for the load cell. From the SETUP screen, align the carousel by selecting CAROUSEL followed by CALIBRATE. Insert the carousel with the 13 small holes facing up into the AFC tower, and adjust the carousel by pressing the minus or plus buttons so that the fluid nozzle is directly above the flush position.

Remove the caps from the equilibrated SEC column, and slide the SEC column into the appropriate column mount. Carefully install it on the AFC tower. Ensure that the radio frequency identification tag on the column faces the AFC, and check that the connection between the column and the valve is secure. Place the waste outlet tubing in a collection container. From the SETUP screen, select Collection Schedule, and set the count to 13, and the size to 0.5 milliliters by pressing the minus or plus buttons.

Leave the buffer volume setting as the default of 2.7 milliliters, and then, close the Collection Schedule window. Select Start Collection from the home screen, and confirm the collection parameters by selecting Yes. Load 13 labeled 1.7-milliliter microcentrifuge tubes with the lids open, and pointing toward the carousel center. Advance the AFC by selecting OK. Then, mount the column reservoir, and advance again by pressing OK.

Select the option to flush the column by pressing, Yes, and add one column volume of 0.2-micrometer-filtered PBS to remove any storage buffer. Once the flush is complete, advance the AFC by pressing OK. Place the carousel cover over the AFC, and press OK. Use a pipette to remove any excess buffer from the column. Bring 3 milligrams of 100R sample to 500 microliters with 1x PBS, and add the sample to the top of the column.

Prepare one column volume of 0.2-micrometer-filtered PBS. Advance the AFC, and allow the sample to run into the frit. Once the sample has fully entered the column, add the PBS prepared earlier to the reservoir. Monitor the run of the AFC while it collects first the void volume, and then, the specified fractions. After the run completes, and the carousel returns to the waste position, remove the cover and the fraction tubes from the carousel.

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