Analysis of Mutant SOD1 Electrophoretic Mobility by Blue Native Gel Electrophoresis; Evidence for Soluble Multimeric AssembliesReportar como inadecuado




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Mutations in superoxide dismutase 1 SOD1 cause familial forms of amyotrophic lateral sclerosis fALS. Disease causing mutations have diverse consequences on the activity and half-life of the protein, ranging from complete inactivity and short half-life to full activity and long-half-life. Uniformly, disease causing mutations induce the protein to misfold and aggregate and such aggregation tendencies are readily visualized by over-expression of the proteins in cultured cells. In the present study we have investigated the potential of using immunoblotting of proteins separated by Blue-Native gel electrophoresis BNGE as a means to identify soluble multimeric forms of mutant protein. We find that over-expressed wild-type human SOD1 hSOD1 is generally not prone to form soluble high molecular weight entities that can be separated by BNGE. For ALS mutant SOD1, we observe that for all mutants examined A4V, G37R, G85R, G93A, and L126Z, immunoblots of BN-gels separating protein solubilized by digitonin demonstrated varied amounts of high molecular weight immunoreactive entities. These entities lacked reactivity to ubiquitin and were partially dissociated by reducing agents. With the exception of the G93A mutant, these entities were not reactive to the C4F6 conformational antibody. Collectively, these data demonstrate that BNGE can be used to assess the formation of soluble multimeric assemblies of mutant SOD1.



Autor: Hilda H. Brown, David R. Borchelt

Fuente: http://plos.srce.hr/



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