Wspólnotowy Serwis Informacyjny Badan i Rozwoju - CORDIS

Development of a blot overlay assay

During the grant period, a blot overlay assay procedure was developed and used to analyse aged muscle fibres. The purified probe was characterized by mass spectrometry, peptide sequencing and immunoblotting. Using a calsequestrin-peroxidase conjugate, it was shown that the major 63-kDa Ca2+-binding protein of the sarcoplasmic reticulum forms complexes with itself, as well as junctin of 26-kDa, triadin of 94-kDa and the ryanodine receptor of 560-kDa. Variations in the relative abundance of the four central elements of excitation-contraction coupling in different fibre types, during myogenesis, ageing and fibre type shifting were reflected by distinct alterations in the calsequestrin overlay binding patterns.

In future application, we will use the overlay procedure for an intraproteomic approach to determine protein-protein interactions within the mature and aged sarcoplasmic reticulum.

Reported by

National University of Ireland
Maynooth, Co.
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