Bradford assay mismeasuring protein seems to be more a consistency issue rather than a methodological problem. I have used this assay and if the calibration curve falls in a range of concentration where the absorbance is linear, the measure should be right. The linearity is very important to have a correct measurment, and it should not be a problem for any kind of sample since you can always use an appropriate dilution. Another thing that came up to me while reading your post is: have you tried to measure the concentration of the protein without lipids, but still following the same protocol? Just brainstorming, but probe sonication can heat the solution and maybe you end up with just less volume caused by evaporation. I really don't known the magnitute of the this effect, so It may be complitely wrong!

For liposome separation, I have encountered recently a similar problem. We tried using Amicon filter units to separate liposome from human protein plasma, but even though there were a lot of protein with MW under the cutoff (100kDa) all proteins were retained. If you are using just specific MW protein this solution may work for you. As an alternative we will try using a SEC column to try separate protein and liposome. Hope this can help you!

I don't have unfortunately any suggestion for the correct ratio, this parameter I think is more empiric than predicted! However, I would suggest you to start with an excess of lipids, fix the amount, and then raise initial cargo amount. In this way you can correlate the EE with the lipid-to-protein ratio. Also, if you have 50% EE, but the initial amount of cargo increase, it increase also the final concentration of cargo, and therefore the amount of absolute encapsulated material. To me, EE is a good process parameter to evaluate the encapsulation mechanism, but lacks of information about how much material is encapsulated.

I hope this point of view may help you, and if you don't agree with something I have written I am opend to discussion (of course!)

Hi !

In the absence of HPLC, one effective alternative is size exclusion chromatography (SEC), which allows separation based on molecular size and can efficiently isolate encapsulated molecules from free ones. Another useful method is dialysis, where free molecules diffuse out through a semi-permeable membrane while vesicles are retained—ensure correct MWCO selection. Additionally, density gradient centrifugation using sucrose or iodixanol gradients may help resolve liposomes from free molecules more cleanly than simple ultracentrifugation. For quantification, fluorescence-based assays using labeled compounds (e.g., FITC-BSA) can help distinguish encapsulated vs. free fractions with higher sensitivity and specificity than Bradford or UV, especially in lipid-rich backgrounds.

For detail Methodology you may refer:

Article Characterization of loaded liposomes by size exclusion chromatography

Thalita Martins Senra Further i would like to tell you..

The efficiency of encapsulation is highly dependent on the lipid-to-drug/protein molar ratio. For liposome systems, typical starting ratios range from 10:1 to 100:1 (lipid:drug or lipid:protein, mol/mol) depending on molecule size, polarity, and charge. For large proteins like BSA, higher lipid content is often necessary due to steric and electrostatic constraints. It is recommended to conduct a loading efficiency curve by varying the lipid concentration while keeping the active constant to identify the saturation point. Additionally, confirm vesicle formation using DLS and ensure proper hydration and extrusion steps are optimized for consistent vesicle size and encapsulation capacity.

Reference for the same:

Article Investigation and Comparison of Active and Passive Encapsula...