I have been producing bacterial OMV and quantifying them using standard BCA protein assay using BSA as standards. However, the protein yield of OMV has been very poor, compared to what mentioned in the literatures. In the protein quantification protocol, I never tried to dissolve the OMV to expose the protein inside. I just make OMV dilutions and lay them in wells then add the substrate substances (all in parallel to the BSA standards) then measure absorbance, according to instructions and calculate the protein content using the standard curve.
Since probably the proteins contents are protected inside the OMV lumen, I'm not sure if this protocol undervalue the OMV protein content so the OMV yield is underestimated???
When I inject this OMV, based on the protein quantification, into mice I see noticeable toxicity. This raises the question of the accuracy of protein quantification. Does anybody recommend a hint on whether I do need to dissolve/rupture the OMV before protein quantification? is there any accurate way to quantify the OMVs?
Hi Sherif Ahmed . From a BCA assay description:
"The BCA assay relies on two reactions. First, peptide bonds reduce Cu2+ ions to Cu1+ . The amount of Cu2+ reduced is proportional to the amount of protein present in the solution. Next, two molecules of bicinchoninic acid chelate with each Cu1+ ion, forming a purple-colored complex that absorbs light at 562 nm."
So, solubilizing proteins, whose peptide bonds would otherwise be inaccessible by a lipid bilayer, should increase sensitivity.
I agree that you should dissolve the OMVs with a detergent if you are measuring the protein content with the BCA assay. If you are using the Bradford assay, the detergent will probably interfere, so the BCA assay would be a better choice.
The use of BSA as the standard may not give an accurate result for OMV proteins because their overall amino acid composition is likely to differ significantly from that of BSA.