Micro-method to isolate and purify amyloid proteins for chemical characterization

The amyloidoses represent a heterogeneous group of disorders characterized by the pathologic deposition as fibrils of at least 20 different precursor molecules. To establish definitively the specific type of amyloid protein c ontained in fibrillar deposits, such material must be extracted, purified, and subjected to amino acid sequence analysis. Heretofore the chemical iden tification of amyloid components has required gram quantities of tissue. Gi ven the often-limited amounts of sample available, e.g., that derived from diagnostic needle biopsies, we have developed a micro-method to isolate and purify amyloid from minute tissue specimens. The procedure involves micro- extraction of the amyloid with subsequent purification by SDS-PA GE, electr oblotting onto PVDF membranes, excision and elution of amyloid protein-rela ted bands, and reversed phase HPLC. Chemical and immunologic studies of iso lated amyloid components have demonstrated the purity achieved with this te chnique and have provided information on the molecular mass, heterogeneity and immunoreactivity of the amyloid. Further, using this methodology, if ha s been possible to obtain sufficient material for amino acid-sequencing and thus to establish unequivocally the chemical and molecular composition of the fibrillar deposits. Our microtechnique has clinical import and also is applicable to analyses of the amyloid found in experimental small animal mo dels of these disorders.