Hi Ece,

When you say that "absorbance values were not significant", do you mean that they were overflow, or that the absorbance was below the lower standard (i.e., near the blank)?

In order to know what is happening, it would be good to know the exosome isolation procedure. Ultracentrifugation or chromatograhpy could result in low recovey, while PEG precipitation could interfere with your BCA assay.

Without further information, I would bet that you could have low protein concentration. You can try the micro BCA assay, which has a lower detection range (from 2-4 ug/ml to 40 ug/ml). Also, Bradford (Comassie Plus) has a methodology to measure down to 1 ug/ml protein, albeit using bigger volume. When I measure protein concentration of exosome preparations, I usually use any of this methods.

Good luck!

Carlos Lopez-Robles

Thank you very much for your detailed response. When I mentioned "absorbance values were not significant," I meant they were below the lower standard (i.e., close to the blank).

I followed the exosome isolation procedure without any notable issues; however, as you pointed out, it could be due to a low protein concentration. I also suspect there may have been an issue with the BSA in my BCA kit. I repeated the experiment by re-diluting the reagents, but encountered the same problem with the absorbance readings. I plan to obtain fresh BSA this week to repeat the test, and if the issue persists, I’ll consider trying the micro BCA assay as you suggested.

Thank you once again for your insights!

Hi Ece,

To check your BSA in the BCA assay, you can evaluate the BSA concentration recovery. Using the calibration curve obtained by the BSA, back-calculate the BSA concentration, using the absorbance value it gives you. That way you can check the linearity of your calibration curve.

Good luck!