Hi Hussein,

Lysosomes is quiet stable protein, however it aggregate at high denaturation concentration GnHCl is preferred.

In addition to DLS and CD you can used relative florescence intensity which will give you an idea of the tertiary structure as well as you can use SEC to determine Monomer loss kinetics.

Dear Ibrahim,

Thanks for your answer. Just to be clear, I already have the aggregated samples of lysozyme. The aim is to test the stability/disassembly of the aggregated sample, and thereby compare the stability of different aggregated samples.

Dynamic light scattering is a very sensitive method for detecting protein aggregation and characterizing the size distribution of the aggregates. It is also quite an easy method, since you only have to put a small sample of the protein in a cuvette, insert it into the instrument, and start the measurement, which takes only a few minutes. You must be careful to avoid any dust or other particulates (other than the protein) in the sample, because the instrument will be thrown off by such things. The sample should not be so aggregated as to be visibly turbid. If the protein is highly aggregated, you may have to dilute the sample to avoid overloading the detector.

A more information-rich method is SEC-MALS, which combines size exclusion chromatography to separate the sample on the basis of size with multiangle light scattering to characterize the size of the particles as they elute.

Light scattering is not the best model to study aggregation, it can be used to study the initial growth of aggregation VS temp or Chemical denaturation.

the best system to study stability is SEC-MALS.

You find out the aggregation window using Dynamic or static light scattering.

Hi Hussein,

may I suggest fluorescence microscopy methods based on RICS?

They may be useful when you are observing inhomogeneous samples where micronscale and nanoscale aggregates coexist. This typically happens in disassembly studies

These methods have been applied in the following works:

1. Fennema Galparsoro D., Zhou X., Jaaloul A., Piccirilli F., Vetri V.*, and Foderà V. Conformational Transitions upon Maturation Rule Surface and pH-Responsiveness of α-Lactalbumin Microparticulates, (2021) ACS Applied Bio Materials 4 (2), 1876-1887. DOI https://doi.org/10.1021/acsabm.0c01541

2. Santangelo, M.G., Foderà, V., Militello, V. and Vetri* V. Back to the oligomeric state: pH-induced dissolution of concanavalin A amyloid-like fibrils into non-native oligomers (2016) RSC Advances, 6 (79), pp. 75082-75091. DOI https://doi.org/10.1039/C6RA16690C

Hi Hussain

You can use the Formic Acid based on this article to evaluate the stability of aggregated forms of lysozyme. We used this protocol to compare the stability of some functioonal amyloid to each other.

Identification of amyloidogenic proteins in the microbiomes of a rat Parkinson's disease model and wild-type rats. Article Identification of amyloidogenic proteins in the microbiomes ...

Kind regards,

Saeid