Myoblots: dystrophin quantification by in-cell western assay for a streamlined development of Duchenne muscular dystrophy (DMD) treatments

Neuropathology and Applied Neurobiology
E Ruiz-Del-YerroV Arechavala-Gomeza

Abstract

New therapies for neuromuscular disorders are often mutation specific and require to be studied in patient's cell cultures. In Duchenne muscular dystrophy (DMD) dystrophin restoration drugs are being developed but as muscle cell cultures from DMD patients are scarce and do not grow or differentiate well, only a limited number of candidate drugs are tested. Moreover, dystrophin quantification by western blotting requires a large number of cultured cells; so fewer compounds are as thoroughly screened as is desirable. We aimed to develop a quantitative assessment tool using fewer cells to contribute in the study of dystrophin and to identify better drug candidates. An 'in-cell western' assay is a quantitative immunofluorescence assay performed in cell culture microplates that allows protein quantification directly in culture, allowing a higher number of experimental repeats and throughput. We have optimized the assay ('myoblot') to be applied to the study of differentiated myoblast cultures. After an exhaustive optimization of the technique to adapt it to the growth and differentiation rates of our cultures and the low intrinsic expression of our proteins of interests, our myoblot protocol allows the quantification of dystrophin a...Continue Reading

References

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Citations

Jan 8, 2019·Journal of Neuromuscular Diseases·Annemieke Aartsma-RusUNKNOWN workshop participants
May 18, 2021·Neuropathology and Applied Neurobiology·Patricia Soblechero-MartínVirginia Arechavala-Gomeza

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