in an affinity column using a corresponding single chain peptide coupled to cnbr activated sepharose 4b pharmacia uppsala sweden as the ligand the specificity of the rabbit antibody was verified by reactivity with appropriate gst fusion protein constructs in western blot analysis data not shown and by cross blocking experiments in which five fold molar excess of the specific peptide but not an irrelevant myotilin peptide residues 199 217 absorbed the reactivity northern blot analysis was performed with a multiple tissue mrna filter clontech laboratories inc palo alto calif using a 32 p labelled 320 bp myotilin cdna fragment which encodes amino acids 369 471 as a probe in vitro translations were performed with a coupled reticulocyte lysate kit promega madison wis using 35 s labelled methionine for detection the templates were full length myotilin and a construct containing amino acids 215 498 myotilin 215 498 in bluescript plasmid vector stratagene for western blotting fresh tissues were homogenized in reducing laemmli buffer equal amounts of protein as estimated by coomassie blue staining were separated in 8 sds page and transferred to nitrocellulose filters schleicher amp schuell gmbh dassel germany the filters were probed with the myotilin antibody or with a control preimmune serum followed by peroxidase conjugated goat anti rabbit igg.