In vitro assay of neurofilament light chain self-assembly using truncated mutants
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- In vitro assay of neurofilament light chain self-assembly using truncated mutants
- Kim, Sung-Kuk; Cho, Sang-Min; Lee, In-Bum; Lee, Young Han; Kang, Jung Hoon; Choi, Jang Hyun; Suh, Pann-Ghill; Chang, Jong-Soo
- Electron microscopy; GST pull-down; Neurofilament light chain; PC12 cells; Self-assembly assay; SW13 cells; Turbidity; Western blotting
- Issue Date
- ELSEVIER SCIENCE BV
- JOURNAL OF NEUROSCIENCE METHODS, v.161, no.2, pp.199 - 204
- Neurofilaments (NFs) are heteropolymers composed of light (NF-L), middle (NF-M), and heavy (NF-H) subunits, present in most neurons. NF-L polymerizes on its own to provide a scaffold on which regular NFs form via the cross-bridging of NF-M or NF-H. To clarify the mechanism of regulation of NF-L self-assembly, we developed an assay using truncated mutant NF-L fused to glutathione-S transferase (GST). Western immunoblotting data show that the GST-fused head-rod domains of NF-L are necessary and sufficient for detecting assembled NF-L. The levels of self-assembled NF-L subunits detected using GST fusion proteins were consistent with those detected by electron microscopy and turbidity assay. Our results collectively imply that GST-fused head-rod domains of NF-L are critical tools for analyzing NF-L self-assembly in vitro.
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