Hi,
we are trying to set up the qEV columns for EV isolation and we are experiencing difficulties with reproducibility. Prior to loading the EVs we concentrate using Amicon filters. Once we have all the fractions we run analyze them by western blot for the presence of CD63. However, we found that CD63 is always present in a completely different fractions and we also realized that the EVs are already going through the column during the first washing step (as these fractions are positive for multiple EV markers). We already tested different columns and we cannot find the solution why EVs would be going through the column so quickly. Does anyone have any idea? Any advice would be greatly appreciated!
Hi Lukas,
from what source are you isolating the EVs? Which pre-separation steps do you perform (e.g., medium-speed centrifugation, filtration)? Which starting volume of your sample do you apply to the Amicon filters before SEC?
Best
Marvin
Hi Marvin,
I am isolating EVs from the conditioned medium supplemented with serum previously depleted of EVs by ultracentrifugation. After collecting the medium I deplete it of cell debris by centrifugation and then I concentrate 6ml to 500 ul with Amicon filter. Then I perform 10 000g centrifugation to deplete the 500 ul of microvesicles and I load all the supernatant (approximately 500 ul) on the column. We use the qEV original/35 nm columns.
Kind regards,
Lukas
Hi Lukas,
I would recommend that you perform the 10,000xg step before Amicon concentration and try if that helps. Also, make sure that your sample input is exactly 500 ul and that all columns are equilibrated to room temperature. Before loading, mix your samples well by pipetting.
You can also check your fractions by Electron Microscopy to see if there are any aggregates.
Did you confirm the Western results by a particle-based technique or an EV flow cytometry method? Also, was the same shift of fractions observed with the total protein amount of the elute?
Best
Marvin
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