We are preparing liposomes encapsulating a protein. We want to calculate the encapsulation efficiency of the protein inside the liposomes. We employ centrifugation to separate large liposomes (the ones we want) from small liposomes plus free protein. We can evaluate both the pellet and the supernatant. We want to disrupt the liposomes (either the large ones or the small ones) and then measure the protein encapsulation by UV spectrophotometry. We haven't achieved a good disruption of liposomes with the detergents we've using. So the question is how we can employ DLS, microscopy and UV spectrophotometry to evaluate if the detergent and the concentration used is actually disrupting the liposomes to produce a traslucid colorless solution that allows the UV analysis of the protein? Thanks a lot.
I did not knew anythings about DLS microscopy. It is interesting. Thanks for mentioned it. So, I studied mechanism of DLS.
A point that I understand is mechanism of DLS depends on size of particle.
Did you highlighting size of your liposomes?
I think it is a key for your problem.
If I am wrong, It will be my pleasure to correcting me.
With best wishes.
Hello Alireza. DLS is employed to evaluate size of the liposomes. Microscopy also can be used about that but I wasn't talking about a combined technique, sorry. Yes, DLS helps to evaluate the size of the liposome, that why we used them to determinate if the detergent concentration used is disrupting the liposomes. But instruments of Malvern, for example, also show correlation function and kcps, I was wondering if one can use those measurements to evaluate liposome disruption.
Hi David.
My means is when your liposomes size be known you can use these method to knowing function of detergents.
Look at "Light scattering experiments
The effect of detergent on Rayleigh light scattering of membranous preparations was measured on a Perkin-Elmer M-PF 44A spectrofluorometer with both monochromators set at 600 nm. In ordinary titration experiments small aliquots of stock detergent solutions (50–100 mg/ml) were added stepwise to 2.5 ml of membrane suspensions (0.5 mg protein/ml of Ca2+-ATPase membranes or 0.25–0.5 mg phospholipid/ml) in a well-stirred cuvette in media containing 10 mM TES (pH 7.4), 100 mM KCl (or 100 mM NaCl in the case of experiments involving SDS), and 0.1 mM CaCl2. Sufficient time was allowed for light scattering to stabilize after each addition. In the case of SDS this required several minutes for Ca2+-ATPase membranes at the critical solubilization levels, and even longer periods (½-1 h) for liposomal membranes..." from " The Mechanism of Detergent Solubilization of Liposomes and Protein-Containing Membranes"
The comma was very important in DLS, microscopy! Interestingly there is a form of DLS microscopy called nanoparticle tracking analysis (NTA). This provides number-based size distributions (as opposed to DLS providing complementary intensity based distributions).. For NTA see: https://www.malvern.com/en/products/technology/nanoparticle-tracking-analysis
Thanks Alireza, I'm gonna search that paper. We tested SDS but maybe the concentration wasn't enough for complete disruption. Dr Alan, do you know how can I use DLS to evalúe disruption of liposomes? I have a Nanozetasizer nanoseries NZ.
You can use the Zetasizer Nano ZS to look at the size changes before and after the liposomes are disrupted. Normally this would be combined with other techniques such as cryo-EM and NTA.
would assume that UV will be not much different for protein free in solution and in 'small liposomes?' Therefore after centrifugation you should be able to calculate encapsulation efficiency for 'large' liposomes. Maybe it could be checked by determination of nitrogen.
Yes! I actually thought about nitrogen determination. Our liposomes have stearylamine but I guess we can measure the difference between blank liposomes and liposomes with protein. I'm going to perform SDS-PAGE and Nitrogen determination, after all. Thank you.
Yes, you can study by DLS, UV and microscopic study. J Oleo Sci. 2014;63(11):1185-93. Epub 2014 Oct 21
DLS technique can give informations about the size distribution and the polydispersity of your liposomes. Thus, i think that you should perform measurments of liposomes size distribution before and after adding surfactant...
Ahmed, I did that, but what if I see no changes? Does it mean that I need to increase the concentration of the detergent?
Exactly, because detergent needs to be more added. In fact, at lows concentrations detergent interacts with loposomes with single molecules. However, for higher concentration, detergent form micelles (self assembly of detergent molecules in spherical form). Thus, it will forms a complex with liposomes. In such case you will see the deferences in DLS results...
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