SEM / sample preparation / protein aggregation
This story started with one of our projects that required protein based biocatalyst that can promote nucleophilic catalysis. Eventually, we found that Lysozyme C clicks all the check-boxes in our list of requirements (RSC Adv. 2016, 6, 208-211). Interestingly, the active catalyst was a lower order assembly of the protein. The monomer and the higher order assembly were found to be 3-4 folds less active. The assembly was probed by intrinsic and extrinsic fluorimetric approaches for the published article.
This blog intend to compare and highlight the data from dynamic light scattering (DLS) and scanning electron microscopy (SEM). It was expected that the two techniques will give different results. However, it is the magnitude of difference (650 times) that is worth noting.
Figure. Image 1 – DLS of 2.5 mM Lysozyme C sample. Image 2 – FE-SEM of a 2.5 mM Lysozyme C (drop casted, vacuum dried and gold coated).
The change in order of assembly is related to sample preparation in most of the examples of electron microscopy. A few tricks and techniques have been able to reduce (but not eliminate) this difference but not without perturbing the assembly. We are eager to see what happens with cryo-EM when the sample is prepared by flash-freeze method.
