title: Detection of Diverse and High Molecular Weight Nesprin-1 and Nesprin-2 Isoforms Using Western Blotting.
abstract: Heavily utilized in cell and molecular biology, western blotting is considered a crucial technique for the detection and quantification of proteins within complex mixtures. In particular, the detection of members of the nesprin (nuclear envelope spectrin repeat protein) family has proven difficult to analyze due to their substantial isoform diversity, molecular weight variation, and the sheer size of both nesprin-1 and nesprin-2 giant protein variants (>800 kDa). Nesprin isoforms contain distinct domain signatures, perform differential cytoskeletal associations, occupy different subcellular compartments, and vary in their tissue expression profiles. This structural and functional variance highlights the need to distinguish between the full range of proteins within the nesprin protein family, allowing for greater understanding of their specific roles in cell biology and disease. Herein, we describe a western blotting protocol modified for the detection of low to high molecular weight (50-1000 kDa) nesprin proteins.
authors.affiliation: School of Biological and Biomedical Sciences, University of Durham, South Road, Durham, DH1 3LE, UK.
authors.forename: James
authors.initials: J
authors.lastname: Carthew
authors.affiliation: School of Biological and Biomedical Sciences, University of Durham, South Road, Durham, DH1 3LE, UK. iakowos.karakesisoglou@durham.ac.uk.
authors.forename: Iakowos
authors.initials: I
authors.lastname: Karakesisoglou
mesh_terms: D000818:Animals; D015153:Blotting, Western / Q000379:methods; D002460:Cell Line; D003598:Cytoskeletal Proteins; D006801:Humans; D008840:Microfilament Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008970:Molecular Weight; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020033:Protein Isoforms
chemical_list: D003598:Cytoskeletal Proteins; D008840:Microfilament Proteins; D009419:Nerve Tissue Proteins; D009687:Nuclear Proteins; D020033:Protein Isoforms; C415465:SYNE1 protein, human; C485904:SYNE2 protein, human
keywords: LINC complex; Nesprins; Nuclear envelope; Polyvinylidene difloride; SDS-PAGE; SYNE; Western blotting