Why do so many dementia treatments fail? Questioning mouse models of tau accumulation bostonchildrens
-labeled arginine . Afterward, the expressed Tau standard was dephosphorylated using Lambda Protein Phosphatase according to the manufacturer’s instructions. Subsequent purification was performed using Ni-Sepharose beads .Frozen tissue samples were homogenized in 5 volumes TBS buffer , protease inhibitor cocktail and Trichlostatin A ), using a Precellys tissue homogenizer . To separate the insoluble Tau species from soluble Tau sarkosyl fractionation was performed on the homogenate.
Before Liquid chromatography-Selective Reaction Monitoring analysis, part of the resuspended sample was spiked with additional peptides. For the mouse models these included the light FLEX-peptide SENLYFQGDISR , the heavy 0N Tau specific peptide STPTAEAEEAGIGDTPSL[+ 7]EDEAA[+ 4]GHVTQA[+ 4]R as well as the peptides carrying respective mutation . Human samples were spiked with heavy 0N, 1N , 3R and P301L Tau specific peptides .
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