I've isolated exosomes from 5ml of cell culture media using ExoQuick TC PLUS kit. I got a visible pellet that was resuspended in the "resuspension buffer" and then purified with the microbeads.
First, I tried to quantify proteins by BCA. I applied the "pure" exosome solution (25 ul in duplicates) and a diluted 1:10 in PBS (resulted in no signal in the end, I got negative values!).
I obtained very low concentration (between 16 and 38 µg/mL).
Then, I sonicated 100µL of my samples and quantified them again by BCA. The concentration improved just a little bit (47 and 61 µg/ml). And this is not enough to do a WB or any downstream application.
The next attempt would be try RIPA buffer, but the exosomes are already in solution.
The problem is that I'm spending my samples only to quantify them.
Do you think that, although the pellet is visible, I'm not obtaining exosomes enough?
I would like to increase the initial medium volume, but this means to scale up the Exoquick reagent and this is financially limiting in the moment.
Is there a method to concentrate the medium before adding Exoquick reagent?
Any advice or tips are welcome!
Thank you
Hi Camila,
Although is a common practice to quantify exosomes using standard protein quantification methods, it is actually incorrect because exosomes are particles. There are three available methods to quantify exosome particles:
1- Electron microscopy (E.M): allow you to determine particle size and therefore distinguish between exosomes and other vesicles, .
2- Nanosight:it is a special optical microscopy adapted to quantify small particles like exosomes (www.nanosight.com)
3- ExoELISA (System Biosciences): this is an ELISA kit especific to quantify exosome particles. I personally tested and works wonderfully, it is particularly recommended if you do not have access to EM or nanosight.
You will find more information in the vendor's website:
http://www.systembio.com/microrna-research/exosome-antibody/elisas/
I hope this helps!...good luck! :-)
Maria Lucrecia Alvarez
https://www.researchgate.net/post/How_can_I_quantify_exosomes_ug_ml_after_purification_without_performing_protein_extraction10
Hi
Exosome purification for a high yield pellet is recommended by ultra-centrifuge at the first place.
https://www.ncbi.nlm.nih.gov/pubmed/25820723
for protein quantification: although BCA is a sensitive method but if you are lysing your pellet with a lysis buffer that contained no detergent, the bradford method is the best choice. BCA interferes with antioxidants and in contrast no interference with detergents. Remember that protease inhibitor cocktails would also be a solution and significantly improve your concentration.
Best
Hi Camila,
Although is a common practice to quantify exosomes using standard protein quantification methods, it is actually incorrect because exosomes are particles. There are three available methods to quantify exosome particles:
1- Electron microscopy (E.M): allow you to determine particle size and therefore distinguish between exosomes and other vesicles, .
2- Nanosight:it is a special optical microscopy adapted to quantify small particles like exosomes (www.nanosight.com)
3- ExoELISA (System Biosciences): this is an ELISA kit especific to quantify exosome particles. I personally tested and works wonderfully, it is particularly recommended if you do not have access to EM or nanosight.
You will find more information in the vendor's website:
http://www.systembio.com/microrna-research/exosome-antibody/elisas/
I hope this helps!...good luck! :-)
Maria Lucrecia Alvarez
https://www.researchgate.net/post/How_can_I_quantify_exosomes_ug_ml_after_purification_without_performing_protein_extraction10
- Badges
- Science topic