TY  - JOUR
TI  - Isolation and Imaging of His- and RFP-tagged Amyloid-like Proteins from Caenorhabditis elegans by TEM and SIM
AU  - Stephens, Amberley D.
AU  - Lu, Meng 
AU  - Kaminski Schierle, Gabriele S.
VL  - 9
IS  - 21
PY  - 2019
DA  - 2019/11/05
SP  - e3408
C1  - Bio-protocol 2019;9:e3408
DO  - 10.21769/BioProtoc.3408
UR  - https://doi.org/10.21769/BioProtoc.3408
AB  - 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.
KW  - His-tag
KW  - RFP-tag
KW  - Amyloid
KW  - Aggregates
KW  - Structured illumination microscopy
KW  - Transmission electron microscopy
KW  - RHO-1
KW  - KIN-19
JF  - Bio-protocol
SN  - 2331-8325
PB  - Bio-protocol LLC.
BIO101  - False