Team:Lethbridge/Experiments
Mass Spectrometry
Mass spectroscopy is a quantitative analysis method that is based on the mass and the charge of the molecule in question. The sample is analyzed by first being transferred to the gas phase which is passed through the analyzer. The signals from the analyzer then sorts the sample ions of fragments by mass and charge (Urban, 2016). From there samples can be identified and formed into recognizable species. This technique is highly specific and selective, making it a great characterization tool for our PNC toolkit. It will be able to prove that the sequences we have used to form our constructs match when comparing this to the mass spectroscopy data we recieve Our mass spectroscopy data for the P22 capsid can be found here.
Analytical Ultracentrifugation
AUC is a way for analyzing macromolecules and interactions quantitatively. There are many advantages of this technology as the is able to work with samples in solution. This technology makes use of three main analytical features: Fluorescence, absorbance and interference. Fluorescence allows for analysis of complex mixtures and interactions of proteins giving distinct data. These techniques coupled with ultracentrifugation can also aid analysis of macromolecular interactions, sizes of complexes/individual molecules and how it travels through solutions without the need for tags or other types of modifications (Cole et al., 2009). In order to highly characterize our protein nano compartments we are using AUC to look at size, capsid formation and how these PNC interact with their cargos. This data will inform our project in terms of encapsulation kinetics and the stoichiometry of PNC to cargo.
This technology is versatile and highly specific which is perfect for characterizing our PNC toolkit, especially our novel PNC, the Arc minimal construct that is derived from HIV homology. Results on our AUC experiment with Arc minimal can be found here. The AUC works by running the samples at a certain temperature and centrifugal force, much like a regular ultracentrifuge. The integrated analysis tools take in the data over time, giving dynamic data as the sample moves through solution.
Figure . Schematic of analytical ultracentrifugation (Wilkinson Group, Universite de Montreal)
Transmission Electron Microscopy (TEM)
Transmission electron microscopy is an advanced form of microscopy used for resolving very small molecular structures or cells (Winey et al., 2014). Due to the inability for structures seen by the naked eye, TEM is crucial for visualizing our ability to form capsids and or see encapsulation of cargos. TEM works by passing an electron beam through a thin layer of sample trapped on a plastic surface which then creates a contrast image formed by the absorbed and scattered light patterns from the beam (Winey et al., 2014). The high amount of resolution and simple methods of sample preparation makes this technology an easy way to visualize and confirm capsid formation of our constructs. Our TEM results for Arc minimal and P22 with cargo SPCas9 can be found here.