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 contained in fibrillar deposits, such material must be extracted, purified, and subjected to amino acid sequence analysis. Heretofore, the chemical identification of amyloid components has required gram quantities of tissue. Given 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-PAGE, electroblotting onto PVDF membranes, excision and elution of amyloid protein-related bands, and reversed phase HPLC. Chemical and immunologic studies of isolated amyloid components have demonstrated the purity achieved with this technique and have provided information on the molecular mass, heterogeneity, and immunoreactivity of the amyloid. Further, using this methodology, it has 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 models of these disorders.
Kaplan
B
B
Heller Institute of Medical Research, Chaim Sheba Medical Center, Tel Hashomer, Israel.
Murphy
C L
CL
Ratner
V
V
Pras
M
M
Weiss
D T
DT
Solomon
A
A
D000595:Amino Acid Sequence; D000682:Amyloid / Q000737:chemistry / Q000302:isolation & purification*; D017772:Amyloid Neuropathies / Q000473:pathology; D000686:Amyloidosis / Q000473:pathology*; D015152:Blotting, Northern; D002850:Chromatography, Gel / Q000379:methods; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D010505:Familial Mediterranean Fever / Q000473:pathology*; D006801:Humans; D008099:Liver / Q000737:chemistry; D012680:Sensitivity and Specificity; D013154:Spleen / Q000737:chemistry
D000682:Amyloid