Use of immunoblot (Western blot) in clinical laboratory for the detection of dystrophin, dysferlin and calpain 3: three relevant proteins in hereditary myopathies diagnosis

Abstract

The absence and/or dysfunction of three constitutive human muscle proteins: dystrophin (UniProtKB - P11532) in the case of dystrophinopathies, and calpain 3 and dysferlin (UniProtKB - P20807 and UniProtKB - O75923, respectively), in limb-girdle muscular dystrophies or LGMD, play a key role in the etiology of these hereditary myopathies. Confirming their presence or absence in muscular biopsies is essential for a correct diagnosis of the disease and underlines the need for a sensitive and specific technique to detect these proteins in the clinical laboratory. To develop a Western blot protocol to be used in the clinical laboratory for the detection and identification of dystrophin, dysferlin and calpain 3 in muscle biopsies. Approximately 20 - 50 mg of muscular biopsies in Urea and SDS containing buffer were analyzed in PAGE/SDS minigels, followed by electrotransference to nitrocellulose membranes, incubation with monoclonal antibodies (MAbs) specific for each of the three mentioned proteins and development with either chromogenic or chemiluminescence reagents. We showed a protocol suitable for the analysis of the large molecular-weight proteins dystrophin (430 Kd), dysferlin (220 Kd) and calpain 3 (95 Kd). Identification was based on both the analysis of their molecular weight and the univocal reactivity with specific monoclonal antibodies (MAbs). In the case of calpain 3, the technique detects not only the 95 Kd complete gene product, but also extra polypeptides in the 55 - 60 Kd range, indicative of a functional protease. The convenience of either the chromogenic or chemiluminescence protocol for signal development is discussed.