[5] Microextraction and purification techniques applicable to chemical characterization of amyloid proteins in minute amounts of tissue

Apolipoprotein AI and Transthyretin as Components of Amyloid Fibrils in a Kindred with apoAI Leu178His Amyloidosis

“…The most striking feature of the amyloid fibrils in our proband is that wild-type TTR is present in the deposits. The method used for fibril extraction 15 is a standard procedure for amyloid fibril isolation from small amounts of tissue, and has been validated to identify unequivocally intrinsic protein components of amyloid deposits. According to this, the proteins extracted correspond only to fibrillary material and it is therefore unlikely that the presence of both TTR and full-length apoAI corresponds to nonamyloid protein components absorbed to the amyloid fibrils.…”

“…Fibrils from a 15-mg skin biopsy were extracted as described by Kaplan et al 15 Briefly, the tissue specimen was homogenized manually with a glass rod with 1 ml of ice-cold phosphate-buffered saline (PBS) and centrifuged for 10 minutes at 14,000 g. This procedure was repeated 3 times to remove soluble blood components. The resulting pellet was resuspended in 1 ml of 20% acetonitrile containing 0.1% trifluoroacetic acid.…”

Definition of organ involvement and treatment response in immunoglobulin light chain amyloidosis (AL): A consensus opinion from the 10th International Symposium on Amyloid and Amyloidosis

“…When definitive immunohistochemical typing of amyloid cannot be achieved, specific genetic studies can be performed by polymerase chain reaction to recognize mutations in transthyretin [44,45], fibrinogen, lysozyme [46], and apolipoproteins A-I and A-II. Amyloid has been extracted successfully from fat deposits [47] for direct sequencing of the fibril protein [48]. Mass spectroscopic analysis of the extracted protein has been used successfully as a diagnostic tool [49,50].…”

SELDI-TOF Mass Spectrometry Evaluation of Variant Transthyretins for Diagnosis and Pathogenesis of Familial Amyloidotic Polyneuropathy

“…All studies using human samples were in accordance with the current revision of the Helsinki Declaration. Tissue-deposited ATTR was extracted as previously described (11 ). Recombinant TTRs were expressed and purified as described previously (12 ).…”