Microdissected Samples

"Proteins from the laser capture microdissected samples of amyloid from the eyelid, conjunctiva and orbit were extracted using a modified protocol of the Liquid Tissue MS Protein Prep Kit (Expression Pathology Inc., Gaithersburg, MD). LCM films containing captured protein deposits and control tissue samples were stripped from the caps and incubated with 40 µl extraction buffer (10 mM Tris, pH 7.8,0.1% RapiGest (Waters)) at 95°C for 90 min. Samples were centrifuged at 10.000 × g for 1 min and subsequently cooled on ice. Extracted proteins were incubated with 5 µg porcine trypsin (Sigma) at 37°C for 16 hours, vortexing every 20 min for the first hour. Samples were then centrifuged at 10.000 × g for 5 min, supernatant isolated and incubated with 10 mM DTT at 95°C for 5 min followed by incubation with 30 mM iodoacetamide for 15 min in the dark. Peptides generated fromeach digest were acidified with 5 µl of 5% formic acid and recovered using Stage-tips (Proxeon, Thermo Scientific) . The peptides were eluted with 10 µl 70 % acetonitrile, and subsequently lyophilized before being dissolved in 12 µl 0.1% formic acid. OMICS Group International is one of the leading Open Access Publishers which is publishing 700+ peer-reviewed journals with the support of 50,000+ editorial board members as editorial team and aimed to disseminate the scholarly knowledge to the scientific society. OMICS Group also organizing 3000+ International Scientific Conferences and events yearly all over the world with the support of 1000+ Scientific associations worldwide. Citation: Nielsen NS, Poulsen ET, Klintworth GK, Enghild JJ (2014) Insight into the Protein Composition of Immunoglobulin Light Chain Deposits of Eyelid, Orbital and Conjunctival Amyloidosis. J Proteomics Bioinform S8: 002"

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Last date updated on September, 2024