Abstract
In our recently published paper, we highlight that during normal aging of C. elegans age-dependent aggregates of proteins form and lead to functional decline of tissues. The protocol described here details the isolation of two proteins from C. elegans in their aggregated amyloid-like form, casein kinase I isoform alpha (KIN-19) and Ras-like GTP-binding protein rhoA (RHO-1). We used nickel beads to isolate His-tagged KIN-19 and RHO-1, and thus permitting the isolation of both small and large aggregated or fibrillary forms of the proteins. We characterized their morphology by transmission electron microscopy. We further expressed RFP-tagged proteins and stained them with a fluorescent molecule, thioflavin T, which identifies β-sheet structures, and which is a defining feature of amyloid fibrils. We further applied structured illumination microscopy to determine the level of colocalization between RFP and thioflavin T.
Highlights
[Background] During aging, there is often a dysregulation in protein homeostasis and an accumulation of damaged or non-functional proteins
Screening for proteins most likely to become insoluble upon aging Caenorhabditis elegans (C. elegans) yielded two target proteins, casein kinase I isoform alpha (KIN-19) and Ras-like GTP-binding protein rhoA (RHO-1) (David et al, 2010)
These two proteins were further studied to investigate their aggregation propensity during the ageing of C. elegans, whether they formed amyloid-like structures and whether they correlated with a functional decline (Huang et al, 2019)
Summary
1. Microlance needle 30G x 0.5” (Fisher Scientific Lt, catalog number: 10442014) 2. 1.5 ml microcentrifuge tubes (Fisher Brand, catalog number: FB74031) 3. Transfer membrane PVDF 0.45 μm (Merck Millipore, catalog number: IVPH08100) 6. Goat anti-Mouse IgG (H + L) Secondary Antibody, Alexa Fluor® 647 conjugate (Merck, catalog number: A21235) 15. Radioimmunoprecipitation assay (RIPA) buffer (Merck, catalog number: R0278-50ML) 19. Supersignal West PicoPLUS Chemiluminescent Substrate (Thermo Fisher Scientific, catalog number: 34577) 25. NuPAGETM LDS buffer (4x) (Thermo Fisher Scientific, catalog number: NP0007) 26. NuPAGETM MES SDS Running buffer (20x) (Thermo Fisher Scientific, catalog number: NP0002) 27. NuPAGETM Transfer Buffer (20x) (Thermo Fisher Scientific, catalog number: NP0006) 28.
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
