I am particularly interested in substrates like MeoSuc-FLF-AFC or LLVY-AMC. These are commonly used in vitro with cell extracts to evaluate proteasome activity, but it would be nice to be able to use them in vivo in combination with a fluorescently-labeled proteasome in C. elegans.
Microinjection of cells might work, but that would be a significant increase in lab work as opposed to soaking cells or animals with the compound.
In addition, it would be helpful to use them in fixed C. elegans, but I doubt this would work, as proteasomal proteolysis requires ATP and intact proteasome complex.
I've never tried to use the synthetic fluoregenics in vivo, but I have quite successfully used the fluorescent proteasome substrate GFP-UbG76V in hippocampal slices before (PMID 22726833). Other attractive options that have been used in primary neural culture include GFP-odc, which is ubiquitination independent, and GFPu, which is tagged with the CL1 degron (see PMID 19638347 and 16810255). The only difficulty I have encountered with this approach is balancing the expression levels between too weak for good photobleaching-free imaging, and too high a concentration for changes in proteasome activity to make a dent in the overall fluorescence. Good luck!
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