1. Determining the topology of peroxisomal proteins using protease protection assays
- Author
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Francisco, T, Dias, AF, Pedrosa, AG, Grou, CP, Rodrigues, TA, Azevedo, JE, and Instituto de Investigação e Inovação em Saúde
- Subjects
Endopeptidases/metabolism ,Peroxisomes/metabolism ,Proteolysis ,Proteins/metabolism ,Endopeptidase K/metabolism - Abstract
Protease protection assays are powerful tools to determine the topology of organelle proteins. Their simplicity, together with the fact that they are particularly suited to characterize endogenous proteins, are their major advantages and the reason why these assays have been in use for so many years. Here, we provide a detailed protocol to use with mammalian peroxisomes. Suggestions on how these assays can be controlled, and how to identify some technical pitfalls, are also presented. This work was financed by FEDER–Fundo Europeu de Desenvolvimento Regional, funds through the COMPETE 2020–Operacional Programme for Competitiveness and Internationalization (POCI), Portugal 2020, and by Portuguese funds through FCT–Fundação para a Ciência e a Tecnologia/Ministério da Ciência, Tecnologia e Inovação in the framework of the projects “Institute for Research and Innovation in Health Sciences” (POCI-01-0145-FEDER-007274) and “The molecular mechanisms of peroxisome biogenesis” (PTDC/BEX-BCM/2311/2014), and through Norte 2020—Programa Operacional Regional do Norte, under the application of the “Porto Neurosciences and Neurologic Disease Research Initiative at i3S (NORTE-01-0145-FEDER-000008)”. T.F, A.F.D., C.P.G., and T.A.R. were supported by Fundação para a Ciência e a Tecnologia, Programa Operacional Potencial Humano do QREN and Fundo Social Europeu.
- Published
- 2017