Dear Magdalini

I'm not an expert in exosomes but i worked with bacterial outer membrane vescicles that are quite similar to the exsosomes and the main problem that i faced to quantify the protein in this vescicles using absorbance at 280nm with nanodrop was the high presence of DNA that can induce overestimation in the protein content.

We used the samples just resuspended in PBS that was our isolation buffer. 

We tthan ried to different approaches:

1) Add benzoase to vescicles respospended in PBS and perform concentration/dilution step to remove the DNA

2) use colorimetric assay, as nanoorange (https://www.thermofisher.com/order/catalog/product/N6666) instead abrosbance at 280nm which is less affected by DNA.

and both seems to work quite well also if I prefer the second because less time expensive and more adaptable in multiwell format. 

Of course  to relate nanoorange absorbance and therefore vescicles protein content to vescicles number you need to be also able to count the number of vescicles in your sample at least one time to create a standard reference curve, and for bacterial  vescicles we used the nanosight instrument. 

good luck

Manuele

Definitely not. Measurements of OD at 280 nm will not produce accurate protein estimation. Just like in the case of crude tissue or cell extracts. Extracellular vesicles contain on their surface proteins, RNA, lipids, all of which would contribute to OD at 280 nm. For accurate EV protein measurement I am using routinely MicroBCA kit (ThermoFisher Scientific).

Thank you very much for the advice !!!

I will try and see what data I will get 

Kindest regards 

Magda 

I tried to take absorbance of exosome protein at 290 nm but RIPA buffer was also showing peaks. So it was very difficult to identify protein peaks.

You can use MicrBCA kit (ThermoFisher Scientific) to estimate protein. RIPA in your exosome sample will not interfere with this assay.

You could precipitate all exosomes in an aliquot from your solution using Trichloroacetic acid. Then suspend in a volume you know of buffer and dose your protein content using BCA test or Bradford depending on your suspension buffer. This way you have the entire proteins of your exosomes. Be careful to have a very pure exosome solutions with few contaminated protein because it would interfere with such approach for dosing exosomes. The ISEV is updating the MISEV guidelines. you may want to look into it soon. Sincerely.

Although it may not be accurate, you may be able to measure exosomal protein content by using nanodrop but you have to lyse exosomes (by using RIPA) for it. We routinely use BCA test (as mentioned above) to analyse exosomal protein content without any problem.

I used Bradford reagent to quantify exosomal protein (by using RIPA buffer), it gave good quantity of protein. whereas the exosomal sample without RIPA buffer treatment (i.e. intact exosomes) did not give any protein.

Hi all. I'm trying to quantify protein from urinary exosomes but remaining urea increases the results we get by BCA. Any suggestions of which is the best approach?

Thank you,

BW