Sep 06, 2022
Detection of Tau ubiquitylation
- Itika Saha1,
- F. Ulrich Hartl2,3,
- Mark S. Hipp2,3
- 1Department of Cellular Biochemistry, Max Planck Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany;
- 2Department of Biomedical Sciences of Cells and Systems, University Medical Center Groningen, University of Groningen, Antonius Deusinglaan, 1, 9713 AV Groningen, The Netherlands;
- 3School of Medicine and Health Sciences, Carl von Ossietzky University Oldenburg, 26129 Oldenburg, Germany
Protocol Citation: Itika Saha, F. Ulrich Hartl, Mark S. Hipp 2022. Detection of Tau ubiquitylation. protocols.io https://dx.doi.org/10.17504/protocols.io.3byl4je4rlo5/v1
Manuscript citation:
The AAA+ chaperone VCP disaggregates Tau fibrils and generates aggregate seeds Itika Saha, Patricia Yuste-Checa, Miguel Da Silva Padilha, Qiang Guo, Roman Körner, Hauke Holthusen, Victoria A. Trinkaus, Irina Dudanova, Rubén Fernández-Busnadiego, Wolfgang Baumeister, David W. Sanders, Saurabh Gautam, Marc I. Diamond, F. Ulrich Hartl, Mark S. Hipp bioRxiv 2022.02.18.481043; doi: https://doi.org/10.1101/2022.02.18.481043
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: September 05, 2022
Last Modified: May 31, 2024
Protocol Integer ID: 69589
Keywords: ASAPCRN
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).
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.
5m
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.
1h
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.