Detection of Tau ubiquitylation
Itika Saha, F. Ulrich Hartl, Mark S. Hipp
Abstract
This protocol describes the detection of ubiquitylated Tau from HEK293 cells stably expressing and propagating aggregates of Tau repeat domain fused to YFP (Sanders et al. Neuron, 2014; Saha et al, BioRxiv, 2022).
Steps
Harvest cells from 2 confluent wells of a 6 well plate.
Lyse aggregate-containing cells by vortexing in cold RIPA buffer (Thermo) supplemented with protease inhibitor cocktail (Roche) and DNase. 20 mM N-ethylmalemide should be included in the lysis buffer to inhibit the activity of deubiquitinating enzymes.
Briefly sonicate lysate and centrifuge at 2,000 x g for 5 min to remove cell debris.
Collect supernatant, determine protein concentration and normalize across samples.
Dilute 1 mg protein in a total volume of 600 µL RIPA buffer.
Add 50 µL anti-GFP bead slurry (µMACS GFP Isolation kit, Miltenyi Biotec) to diluted lysate.
Incubate for 1 h at 4 °C in a rotating wheel at 10 rpm.
Before the end of 1 h, place µ-columns (Miltenyi Biotec) in the magnetic field of a µMACS Separator (Miltenyi Biotec) and equilibrate columns by applying 250 µL RIPA buffer. Allow complete flow-through.
Apply cell lysates and beads to µ-columns. Allow complete flow-through.
Wash columns 4 times with 1 mL 0.1% SDS/PBS.
Elute by applying 50 µL pre-heated (95 °C) 1x SDS sample buffer.
Analyze eluates by immunoblotting with antibodies against GFP or ubiquitin.
